Southern Blot

Introduction

A Southern blot is a laboratory method used to detect specific DNA molecules among many other DNA molecules. The technique is named after its inventor, Edward Southern. As a laboratory procedure, Southern blots can be used to analyze an organism’s total DNA, also known as its genome, to identify a specific sequence of interest.

The first step in a Southern blot is to prepare the DNA mixture by breaking it into small fragments using a protein called a restriction enzyme. The mixture of DNA fragments is then separated by size using a technique called gel electrophoresis. After separation, the double-stranded DNA pieces denature or separate into single strands within the gel. The DNA is then transferred from the gel to a transfer membrane. Although this step is what gives the technique the name “southern transfer,” the term is generally used to describe the entire procedure.

Once the transfer is complete, the membrane carries all the bands originally on the gel. The membrane is then treated with a small piece of DNA or RNA called a probe, which has been designed to have a sequence that is complementary to a particular DNA sequence in the sample; this allows the probe to hybridize or bind to a specific DNA fragment on the membrane. Also, the probe has a label, which is usually a radioactive atom or a fluorescent dye. Thus, after hybridization, the probe allows the DNA fragment of interest to be detected among the many different DNA fragments on the membrane.

General procedure for drying

1. Sample homogenization involving DNA/RNA/protein purification that is performed after extraction from a variety of sources, such as cells or tissues.

2. Digest DNA with restriction enzymes into fragments, which is not necessary for RNA (northern blot).

3. Separation of the molecule of interest by membrane electrophoresis, generally on agarose gel for DNA fragments. In the case of RNA samples, they can be separated on an agarose gel in the presence of formaldehyde as a denaturing agent. This is necessary since formaldehyde limits the secondary structures of RNA molecules.

4. Transfer the molecules (DNA/RNA fragments) to a nitrocellulose membrane/nylon membrane from the gel.

5. Prehybridization (Blocking): Washing the nylon membrane with a prehybridization or blocking solution comprising salmon sperm DNA is required to block non-specific DNA interactions and also helps reduce background noise. Alternatively, there are some commercially available blocking buffers, such as PerfectHyb™ Plus buffer, in which salmon sperm DNA is not required for blocking purposes.

6. For probe preparation, a fresh 32P alpha-labelled dCTP-labeled DNA probe is prepared.

7. Hybridization or identification of the molecule that is achieved by incubating the blot with the specific labelled probe.

8. For detection of the probe and the DNA/RNA sequence of interest, the film is exposed to -80°C.

Beginning

Restriction endonucleases, which is enzymes, are used to break DNA into small fragments. These fragments are then separated by electrophoresis. The fragments obtained are then classified according to their size (kDa). Thus, the DNA fragments are transferred to blotting paper where they are incubated with probes. The probes used in Southern blotting can be very selective. They can selectively bind with a resolution of 1 in a million and the characteristics to bind to the intended target fragments.

Necessary materials reagents

  • The buffer used for electrophoresis is TAE or TBE.
  • The preferred agarose should be electrophoresis grade.
  • For DNA staining, ethidium bromide (0.5 µg ml–1, dissolved in H2O) is used. But there are other DNA staining dyes, such as SYBR green, that can replace ethidium bromide for safe handling.
  • 2X and 20X SSC (20X SSC composition includes 3.0 M NaCl and 0.3 M sodium citrate)
  • 6X DNA Loading Buffer consisting of 0.25% Bromophenol Blue, 0.25% Xylene Cyanol FF, and 30% Glycerol in water.
  • Suitable DNA markers of variable molecular weight, also called DNA ladders, are used as reference standards.
  • The prehybridization and hybridization mixture consists of 0.5% SDS, 6X SSC, 5X Denhardt’s solution, and 100 mg ml–1 sheared denatured salmon sperm DNA of yeast tRNA.
  • The widely used Denhardt solution for hybridization is composed of 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin (BSA)
  • Paraffin oil
  • Cellulose nitrate or nitrocellulose membrane filter with uniform porosity. (eg, Millipore 25 HAWP; nylon membranes used for blot protocols are available under various trade names from commercial vendors)
  • RNase A (20 pg ml–1 in 2X SSC) is required for specific cleavage.
  • Restriction enzyme and an appropriate buffer are used.
  • Radioactively labelled RNA as a probe for specific detection where autoradiography is performed.
  • For the detection of RNA labelled with labels such as 3H, 35S, 125I or 14C, 2,5-diphenyloxazole (PPO) in toluene at a concentration of 20% w/vol is required.

Team

1. Transfer of narrow strips of gel can be achieved using three pieces of glass or Plexiglas that are 5 cm × 20 cm in size with a thickness similar to the thickness of the gel.

2. There is a requirement for coarse, dry filter paper (four to five in number) or paper towels 10 cm × 18 cm in size.

3. The hybridization dish has a larger dimension (0.8 mm depth × 2 cm height × ~1 cm length) than the membrane used for hybridization, and the material used to develop the hybridization dish is Perspex (note: several alternative procedures for hybridization follow)

4. Four narrow pieces of Perspex that are similar in thickness to gel. Perspex length is sufficient to surround the gel at a distance of ~3 mm

5. A tray 20–50 mm (approx.) deep and 20 mm (approx.) in length and width larger than the gel

6. A sheet of glass long enough to fit in the pan and narrower to have a 10mm gap on each side

7. Several thick pieces of filter paper have a large area compared to the gel. The length of the filter paper is suitable to cover the glass sheet and can be immersed inside the tray.

8. A moistened piece of nitrocellulose membrane, which has a wider area that can cover the entire gel. The nitrocellulose membrane is placed on top of four strips of Perspex. Wetting of the nitrocellulose membrane is done using 2X SSC.

9. Paper towels that are stacked on top of each other.

10. Apparatus for straining gel.

11. A gel tank is necessary to perform electrophoresis.

12. Power supply is required for all device configurations.

Reagent Configuration

  • DNA: The entire procedure begins using DNA digested with enzymes of varying concentrations that will quantify the optimal concentration of DNA and specify the restriction enzyme to be used. Generally, an amount of 1 µg of DNA derived from clones (eg, from plasmid or bacteriophage clones) is sufficient for plasmids having low copy numbers. We need larger amounts to carry out the separation of complex DNA (eg genomic DNA). The advisable range to consider would be 5 to 10 µg.
  • The electrophoresis buffer used is TAE, which has a composition of 40 mM Tris, 20 mM acetic acid, 1 mM EDTA with a pH range of 7.4 to 8.2, which is normally prepared as a stock concentration of 20X or TBE composed of 89 mM Tris, 2.5 mM EDTA, 89 mM borate, normally made as a 10× stock)
  • TAE is recommended to be better when running gels for a shorter time interval and when recovery of DNA fragments from the gel is to be carried out.
  • TBE is considered to be a better buffer, especially when we have to run the gels for longer than 2 h.

Request

Southern blot is used in various applications. The main use of the Southern blot is to identify a specific DNA in a DNA sample. It is mainly used in the identification of viral infections and certain bacterial infections. In rDNA technology, the Southern blot technique is used to isolate a particular DNA. It is also useful in the study of genetic mutations and rearrangements, this property is used to diagnose neonatal diseases and genetic diseases. Due to the accuracy in DNA identification, this technique is used in phylogenetic studies, paternity and maternity analysis, forensic studies and personal identification.

Southern blotting can be applied to study the structure of a gene or to elucidate restriction enzyme maps. In particular, Southern blotting can be used to identify the methylated sites present for particular genes. This can be implemented by applying restriction nucleases such as MspI and HpaII, which can identify and cleave between identical sequences. The discovery of RFLPs by Southern blotting has aided in the mapping of several genomes that were crucial to map. In the field of immunology, clonal rearrangements of immunoglobulins, as well as T-cell receptor genes that play an important role in eliciting an immune response, can be analyzed by Southern blotting.

Slipped-strand mispairing

Abstract

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following features characterize such sequences: (1) they typically consist of a variety of 1-10 base repeat motifs, but may also include much larger repeats; (2) larger repeating units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped strand mismatch events, together with unequal crossing over, can easily explain all of these features.

The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to be the result of non-random patterns of nucleotide substitution. We argue that the intrahelical process of slipped strand mismatching is much more likely to be the major factor in the initial expansion of short repeat motifs and that, after the initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossover or other interhelical events due to their propensity for mismatching.

Evidence is presented that single base repeats (the shortest possible motifs) are represented by longer series in mammalian introns than would be expected randomly, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Thus, simple repetitive sequences may represent a natural ground state of DNA not selected for coding functions.

Key points

  • Altered gene expression is the result of SSM and, depending on where the increase or decrease in short repeat sequences occurs relative to the promoter, will be regulated at the level of transcription or translation. The result is an ON or OFF phase of a gene or genes.
  • SSM can result in an increase or decrease in the number of short repeat sequences. Short repeat sequences are from 1 to 7 nucleotides and can be homogeneous or heterogeneous repetitive DNA sequences.
  • Transcriptional regulation can occur if the repeats are located in the promoter region at the RNA polymerase binding site, -10 and -35 upstream of the gene(s).
  • SSM induces transcriptional regulation by changing short repeat sequences located outside the promoter. If there is a change in the short repeat sequence, it can affect the binding of a regulatory protein, such as an activator or a repressor.

Key terms

Slipped strand mismatch: a process that produces a mismatch of short repeat sequences between the parent strand and the daughter strand during DNA synthesis.

7.1D: Slipped-Strand Mispairing - Biology LibreTexts

Slipped strand mismatch (SSM) is a process that results in the mispairing of short repeated sequences between the mother and daughter strand during DNA synthesis. This RecA-independent mechanism can occur during DNA replication or DNA repair and can be on the leading or lagging strand and can result in an increase or decrease in the number of short repeat sequences. Short repeat sequences are from 1 to 7 nucleotides and can be homogeneous or heterogeneous repetitive DNA sequences.

Altered gene expression is the result of SSM and, depending on where the increase or decrease in short repeat sequences occurs relative to the promoter, will be regulated at the level of transcription or translation. The result is an ON or OFF phase of a gene or genes. Transcriptional regulation occurs in several ways. One possibility is if the repeats are located in the promoter region at the RNA polymerase binding site, -10 and -35, upstream of the gene(s). The opportunistic pathogen H. influenza has two divergently oriented promoters in the gene shift and hifB fimbriae.

Overlapping promoter regions have TA dinucleotide repeats at -10 and -35 sequences. Via SSM, the TA repeat region can undergo the addition or subtraction of TA dinucleotides, resulting in the reversible ON or OFF phase of hifA and hifB transcription. The second way that SSM induces transcriptional regulation is by changing short repeat sequences located outside of the promoter. If there is a change in the short repeat sequence, it can affect the binding of a regulatory protein, such as an activator or a repressor. It can also give rise to differences in the post-transcriptional stability of the mRNA.

Restriction Fragment Length Polymorphisms (RFLPs)

Abstract

Restriction fragment length polymorphisms, or RFLPs, are differences between individuals in the lengths of DNA fragments cut by enzymes. Restriction enzymes are proteins that cut DNA at short, specific sequences called restriction sites. After cutting a segment of DNA with restriction enzymes, researchers can examine the fragments using a laboratory method called gel electrophoresis, which separates DNA fragments based on their size.

If two individuals have differences in their DNA sequences at particular restriction sites, the restriction enzymes will cut their DNA into fragments of different lengths. There may also be differences in the number of DNA fragments observed between two or more individuals. Gentaur RFLP analysis can be used as a form of genetic testing to see if an individual carries a mutant gene for a disease that runs in their family.

Beginning

Restriction endonucleases are enzymes that cut long DNA into short pieces. Each restriction endonuclease targets different nucleotide sequences in a DNA strand and therefore cuts at different sites. The distance between the cleavage sites of a given restriction endonuclease differs between individuals. Therefore, the length of the DNA fragments produced by a restriction endonuclease will differ between individual organisms and species.

How does it work?

The RFLP is carried out through a series of steps that are briefly described below:

  • Extraction of DNA

To begin with, DNA is extracted from blood, saliva, or other samples and purified.

  • DNA fragmentation

The purified DNA is digested using restriction endonucleases. The recognition sites of these enzymes are generally 4 to 6 base pairs in length. The shorter the recognized sequence, the greater the number of fragments generated from digestion.

For example, if there is a short GAGC sequence that occurs repeatedly in a DNA sample. The restriction endonuclease that recognizes the GAGC sequence cuts the DNA at each repeat of the GAGC pattern. If one sample repeats the GAGC sequence 4 times while another sample repeats it 2 times, the length of the fragments generated by the enzyme for the two samples will be different.

  • Gel electrophoresis

Restriction fragments produced during DNA fragmentation are analyzed by gel electrophoresis. The fragments are negatively charged and can be easily separated by electrophoresis, which separates molecules based on their size and charge. The fragmented DNA samples are placed in the chamber containing the electrophoretic gel and two electrodes. When an electric field is applied, the fragments migrate towards the positive electrode. Smaller fragments move faster through the gel, leaving larger ones behind, and therefore the DNA samples separate into distinct bands on the gel.

  • Band Visualization

The gel is treated with luminescent dyes to make the DNA bands visible.

RFLP Applications

RFLP has been used for various genetic analysis applications since its invention.

Some of these key RFLP applications are listed below:

  • Determine the status of genetic diseases such as Cystic Fibrosis in an individual.
  • To determine or confirm the origin of a DNA sample, such as in paternity tests or criminal investigations.
  • In genetic mapping to determine recombination rates showing the genetic distance between loci.
  • To identify a carrier of a disease-causing mutation in a family.

RFLP Disadvantages

1. Since its invention, RFLP has been a widely used genome analysis technique in forensic science, medicine, and genetic studies. However, it has become almost obsolete with the advent of relatively simple and less expensive DNA profiling technologies, such as polymerase chain reaction (PCR).

2. The RFLP procedure requires numerous steps and takes weeks to obtain results, while techniques such as PCR can amplify target DNA sequences in a few hours.

3. In addition, RFLP requires a large DNA sample, the isolation of which can be a laborious and time-consuming process. In contrast, PCR can amplify minute amounts of DNA in a matter of hours.

4. Due to numerous reasons like these, the PCR technique has largely replaced RFLP in most applications that require DNA sequencings, such as paternity testing or forensic sample analysis.

5. Furthermore, the identification of single nucleotide polymorphisms in the Human Genome Project has nearly replaced the need for RFLP in disease state analysis.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

Molecular strategies are more and more being utilized to stained cytology slides for the analysis of neoplastic and infectious ailments. Such strategies for the identification of fungi from stained cytology slides haven’t but been evaluated. This research aimed to evaluate the diagnostic accuracy of direct (with out nucleic acid isolation) panfungal polymerase chain response (PCR) adopted by sequencing for identification of fungi and oomycetes on stained cytology slides from canines, cats, horses, and different species. Thirty-six circumstances had been recognized with cytologically identifiable fungi/oomycetes and concurrent identification through fungal tradition or immunoassay.

Twenty-nine controls had been recognized with no cytologically or histologically seen organisms and a concurrent adverse fungal tradition. Direct PCR concentrating on the inner transcribed spacer area adopted by sequencing was carried out on one cytology slide from every case and management, and the sensitivity and specificity of the assay had been calculated. The sensitivity of the panfungal PCR assay carried out on stained cytology slides was 67% total, 73% excluding circumstances with oomycetes, and 86% when contemplating solely slides with plentiful fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from management slides with no seen fungus and adverse tradition outcomes.

Workplace-collected blood spots deposited on filter paper had been analysed with multiplexed affinity-based protein assays and located to be appropriate for proteomics evaluation. The protein extension assay (PEA) was used to characterize 92 proteins utilizing 1.2 mm punches in repeated samples collected from 20 employees. Overall, 97.8% of the samples and 91.3% of the analysed proteins handed high quality management. Both inside and between spot correlations utilizing six replicates from the identical particular person had been above 0.99, suggesting that comparable ranges are obtained from a number of punches from the identical spot and from consecutive spots.

Protein ranges from dried blood and moist serum from the identical people had been in contrast and the bulk of the analysed proteins had been discovered to be considerably correlated. These outcomes open up for simplified pattern assortment of blood in discipline circumstances for proteomic evaluation, but additionally spotlight that not all proteins might be robustly measured from dried complete blood. Direct panfungal PCR is succesful of offering genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay needs to be carried out on slides with seen fungi and interpreted along side morphologic evaluation by a medical pathologist.

Paper-based loop-mediated isothermal amplification and lateral stream (LAMP-LF) assay for identification of tissues of cattle origin

The current research was made with the aims of growth and standardization of cattle particular paper-based loop-mediated isothermal amplification cum lateral stream assay (LAMP-LFA), as a Point-of-care check (POCT) for identification of tissue of cattle origin. The parts of standardized LAMP response using cattle particular primer units had been lyophilized over paper buttons, recognized finest because the provider of LAMP reagents. Based on possible LAMP amplicon, a pair of probes was designed, tagged and its hybridization with the amplified product of paper LAMP response was optimized. The parts of lateral stream assay for detection of probe hybridized LAMP merchandise had been standardized. Analysis of profitable amplification was made by utilizing HNB dye, LAMP-LFA strip, and likewise by the standard ladder-like sample on gel electrophoresis.

The assay was discovered extremely particular for cattle with an analytical sensitivity of 0.1 pg of absolute DNA. Laboratory validation carried out on samples from completely different people of cattle, coded samples, binary meat admixture, and heat-processed cattle tissues substantiated the accuracy of the assay. Comparison with pre-standardized species-specific PCR assay taken as gold requirements revealed 100% conformity. The discipline utility of the developed assay was additional established by its compatibility with the business package eliminating the prolonged DNA extraction step and storage stability of LAMP reagent provider buttons for Four months below refrigeration. Thus, the developed assay succesful of the end result inside Three h in resource-limited settings can be utilized as POCT for identification of tissue of cattle origin.

 Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

High-Speed Lens-Free Holographic Sensing of Protein Molecules Using Quantitative Agglutination Assays

Accurate, cost-effective, easy-to-use, and point-of-care sensors for protein biomarker ranges are necessary for illness diagnostics. A cheap and compact readout method that has been used for a number of diagnostic functions is lens-free holographic microscopy, which offers an ultralarge discipline of view and submicron decision when it’s coupled with pixel super-resolution strategies. Despite its potential as a diagnostic method, lens-free microscopy has not beforehand been utilized to quantitative protein molecule sensing in answer, which might simplify sensing protocols and finally allow measurements of binding kinetics in physiological circumstances.

Here, we sense interferon-γ (an immune system biomarker) and NeutrAvidin molecules in answer by combining lens-free microscopy with a one-step bead-based agglutination assay, enabled by a customized high-speed light-emitting diode (LED) array and automatic picture processing routines. We name this a quantitative large-area binding (QLAB) sensor.

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  • 100 tests
  • 25 tests

Prosi Silver Staining Kit for 25 mini gel

S3200-020 1kit
EUR 361

Live-or-Dye UV/448 Fixable Viability Staining Kit (200 assays)

32002 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Live-or-Dye 405/452 Fixable Viability Staining Kit (200 assays)

32003 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Live-or-Dye 488/515 Fixable Viability Staining Kit (200 assays)

32004 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Live-or-Dye 568/583 Fixable Viability Staining Kit (200 assays)

32005 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Live-or-Dye 594/614 Fixable Viability Staining Kit (200 assays)

32006 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Live-or-Dye 640/662 Fixable Viability Staining Kit (200 assays)

32007 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Live-or-Dye 750/777 Fixable Viability Staining Kit (200 assays)

32008 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Live-or-Dye 405/545 Fixable Viability Staining Kit (200 assays)

32009 1kit
EUR 283
Description: Minimum order quantity: 1 unit of 1kit

Cell Navigator™ Lysosome Staining Kit *Deep Red Fluorescence*

22659 500 Tests
EUR 176

Cell Navigator™ Mitochondrion Staining Kit *Deep Red Fluorescence*

22669 500 Assays
EUR 219

Caspase Screening Kit

K2056-25 25 assays
EUR 279

Human Active caspase 3 ELISA kit

E01A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Human Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Human Active caspase 3 ELISA kit

E01A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Human Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rat Active caspase 3 ELISA kit

E02A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Rat Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rat Active caspase 3 ELISA kit

E02A0015-48 1 plate of 48 wells
EUR 520
Description: A competitive ELISA for quantitative measurement of Rat Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rat Active caspase 3 ELISA kit

E02A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Rat Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rabbit Active caspase 3 ELISA kit

E04A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Rabbit Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rabbit Active caspase 3 ELISA kit

E04A0015-48 1 plate of 48 wells
EUR 520
Description: A competitive ELISA for quantitative measurement of Rabbit Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Rabbit Active caspase 3 ELISA kit

E04A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Rabbit Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Mouse Active caspase 3 ELISA kit

E03A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Mouse Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Mouse Active caspase 3 ELISA kit

E03A0015-48 1 plate of 48 wells
EUR 520
Description: A competitive ELISA for quantitative measurement of Mouse Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Mouse Active caspase 3 ELISA kit

E03A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Mouse Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Pig Active caspase 3 ELISA kit

E07A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Porcine Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Pig Active caspase 3 ELISA kit

E07A0015-48 1 plate of 48 wells
EUR 520
Description: A competitive ELISA for quantitative measurement of Porcine Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Pig Active caspase 3 ELISA kit

E07A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Porcine Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Monkey Active caspase 3 ELISA kit

E09A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Monkey Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Monkey Active caspase 3 ELISA kit

E09A0015-48 1 plate of 48 wells
EUR 520
Description: A competitive ELISA for quantitative measurement of Monkey Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Monkey Active caspase 3 ELISA kit

E09A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Monkey Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Goat Active caspase 3 ELISA kit

E06A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Goat Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Goat Active caspase 3 ELISA kit

E06A0015-48 1 plate of 48 wells
EUR 520
Description: A competitive ELISA for quantitative measurement of Goat Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Goat Active caspase 3 ELISA kit

E06A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Goat Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Dog Active caspase 3 ELISA kit

E08A0015-192T 192 tests
EUR 1270
Description: A competitive ELISA for quantitative measurement of Canine Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Dog Active caspase 3 ELISA kit

E08A0015-48 1 plate of 48 wells
EUR 520
Description: A competitive ELISA for quantitative measurement of Canine Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

Dog Active caspase 3 ELISA kit

E08A0015-96 1 plate of 96 wells
EUR 685
Description: A competitive ELISA for quantitative measurement of Canine Active caspase 3 in samples from blood, plasma, serum, cell culture supernatant and other biological fluids. This is a high quality ELISA kit developped for optimal performance with samples from the particular species.

JC-1 Mitochondrial Membrane Potential Detection Kit (25 assays)

30001-T 1kit
EUR 127
Description: Minimum order quantity: 1 unit of 1kit

human MMP-9 activity assay kit 96-assays

QZBmmp9H 1 plate 96 assays
EUR 501

mouse MMP-9 activity assay kit 96-assays

QZBmmp9M 1 plate 96 assays
EUR 501

Caspase 9 antibody

70R-49474 100 ul
EUR 244
Description: Purified Polyclonal Caspase 9 antibody

Caspase 9 antibody

70R-49475 100 ul
EUR 244
Description: Purified Polyclonal Caspase 9 antibody

Caspase 9 antibody

70R-51476 100 ul
EUR 244
Description: Purified Polyclonal Caspase 9 antibody

Caspase 9 antibody

70R-33457 100 ug
EUR 327
Description: Rabbit polyclonal Caspase 9 antibody

Caspase 9 antibody

70R-33459 100 ug
EUR 327
Description: Rabbit polyclonal Caspase 9 antibody

Caspase 9 antibody

70R-33461 100 ug
EUR 327
Description: Rabbit polyclonal Caspase 9 antibody

Caspase 9 Antibody

AF6348 200ul
EUR 304
Description: Caspase 9 Antibody detects endogenous levels of total Caspase 9.

Caspase 9 Antibody

ABF6348 100 ug
EUR 438

anti-Caspase 9

YF-PA23361 50 ul
EUR 334
Description: Mouse polyclonal to Caspase 9

Caspase 9 protein

30R-2406 25 units
EUR 375
Description: Purified recombinant Human Caspase 9 protein

Caspase 9 protein

30R-2407 50 ug
EUR 581
Description: Purified recombinant Human Caspase 9 protein

Caspase-9 Antibody

3016-100
EUR 338

Caspase-9 Antibody

3016-30T
EUR 146

Caspase 9 antibody

20R-1425 100 ug
EUR 673
Description: Rabbit polyclonal Caspase 9 antibody

Caspase 9 antibody

20R-1468 100 ug
EUR 673
Description: Rabbit polyclonal Caspase 9 antibody

Caspase 9 antibody

20R-1472 100 ug
EUR 781
Description: Rabbit polyclonal Caspase 9 antibody

Caspase 9 antibody

20R-1557 100 ug
EUR 673
Description: Rabbit polyclonal Caspase 9 antibody

Caspase 9 antibody

20R-2497 50 ug
EUR 281
Description: Rabbit polyclonal Caspase 9 antibody

The high-speed gentle supply offers, for the primary time, pixel super-resolved imaging of >104 2 μm beads in answer present process Brownian movement, with out vital movement blur. The automated picture processing routines allow the counting of particular person beads and clusters, offering a quantitative sensor readout that relies upon on each bead and analyte concentrations. Fits to the chemical binding idea are offered. For NeutrAvidin, we discover a restrict of detection (LOD) of <27 ng/mL (450 pM) and a dynamic vary of 2-Four orders of magnitude. For mouse interferon-γ, the LOD is <Three ng/mL (200 pM) and the dynamic vary is at the very least Four orders of magnitude. The QLAB sensor holds promise for point-of-care functions in low-resource communities and the place protocol simplicity is necessary.

Lysosome Targeting RedGreen-assay: Selective Autophagy Sensing Assay for Mammalian Cells

Lysosome Targeting RedGreen-assay: Selective Autophagy Sensing Assay for Mammalian Cells

The means of autophagy is a necessary mobile mechanism, required to take care of basic cell well being by the elimination of dysfunctional organelles, such because the ER, peroxisomes and mitochondria, in addition to protein aggregates, and micro organism. Autophagy is an especially dynamic course of, and instruments are continually being developed to check the varied steps of this course of. This protocol particulars a technique to check the top steps of autophagy-lysosomal fusion and the formation of the autolysosome. Many strategies have been used to check the varied steps of the autophagy course of.

Here we describe the RedGreen-assay (RG-assay), an immunofluorescence-based method used to visualise the concentrating on of substrates to the autolysosome in reside cells. This method takes benefit of the low lysosomal pH and over-expression of a tandem GFP-mCherry tagged protein focused to an organelle of curiosity. While within the impartial cytosol or autophagosome, each GFP and RFP will fluoresce. However, inside the autolysosome, the GFP sign is quenched as a result of low pH surroundings and the RFP emission sign will predominate. This method is quickly quantifiable and amenable to excessive throughput experiments. Additionally, by tagging the GFP-RFP tandem fluorescent protein with organelle particular concentrating on sequences, it may be used to measure a variety of substrates of autophagy.

Quantifying the ratio of alternatively spliced mRNA variants of genes with identified various splicing variants is extremely related for many purposes. Herein, we describe the validation of a quantitative PCR design for the simplified quantification of identified mRNA splice variants. The assay makes use of a single-common primer pair, twin probe design for the willpower of splicing variants in a single nicely configuration. We used murine XBP-1 splicing variants, XBP-1S and XBP-1U, to validate and show the efficiency traits of this method. Using artificial XBP-1S and XBP-1U cDNA in addition to cDNA synthesized from mouse beta-cell line MIN6, we established the efficiency parameters and dynamic vary of the assay. Reliable quantification of each variants at various focus gradients was proven.

Platelet Isolation and Activation Assays

Platelets regulate hemostasis and are the important thing determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets flow into in an inactive state, however develop into activated in response to wreck to the endothelium, which exposes thrombogenic materials resembling collagen to the blood circulate. Activation leads to plenty of responses, together with secretion of soluble bioactive molecules through the discharge of alpha and dense granules, activation of membrane adhesion receptors, launch of microparticles, and externalization of phosphatidylserine.

These processes facilitate agency adhesion to websites of damage and the recruitment and activation of different platelets and leukocytes, leading to aggregation and thrombus formation. Platelet activation drives the hemostatic response, and likewise contributes to pathogenic thrombus formation. Thus, quantification of platelet-associated responses is vital to many pathophysiologically related processes. Here we describe protocols for isolating, counting, and activating platelets, and for the fast quantification of cell floor proteins utilizing circulate cytometry.

Extracellular vesicles (EVs) are produced by all domains of life together with Bacteria, Archaea and Eukarya. EVs are important for mobile physiology and comprise assorted cargo: virulence components, cell wall transforming enzymes, extracellular matrix parts and even nucleic acids and metabolites. While varied protocols for isolating EVs have been established for mammalian cells, the sphere is actively growing instruments to check EVs in different organisms.

In this protocol we describe our strategies to carry out density gradient purification of EVs in bacterial cells, permitting for separation of EV subpopulations, adopted by safety assays for EV cargo characterization. Furthermore, we devised a protocol which includes a fluorescent conjugate of fatty acids into EVs, the primary to permit reside-cell EV monitoring to look at launch of EVs, together with throughout an infection of mammalian cells by pathogenic micro organism. These protocols are highly effective instruments for EV researchers as they allow the statement of EV launch and the research of the mechanisms of their formation and launch.

 Lysosome Targeting RedGreen-assay: Selective Autophagy Sensing Assay for Mammalian Cells

Ex vivo Drosophila Wing Imaginal Disc Culture and Furin Inhibitor Assay

Furin is an evolutionarily conserved proprotein convertase (PC) household enzyme with a broad vary of substrates which might be important for developmental, homeostatic, and illness pathways. Classical genetic approaches and in vitro biochemical or cell organic assays recognized that precursor types of most development issue household proteins are processed by Furin. To quantitatively assess the potential function of Furin in cleaving and modulating intercellular dispersion of a Drosophila signaling protein, we developed a easy assay by combining genetics, ex vivo organ tradition, pharmacological remedy, and imaging analyses.

Caspase-9 Colorimetric Assay Kit

K119-200
EUR 620

Caspase-9 Colorimetric Assay Kit

K119-400
EUR 958

Caspase-9 Colorimetric Assay Kit

K2019-100 100 assays
EUR 529

Caspase-9 Colorimetric Assay Kit

K2019-200 200 assays
EUR 696

Caspase-9 Colorimetric Assay Kit

K2019-400 400 assays
EUR 1156

Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay Kit (25 assays)

30011-1 1KIT
EUR 204
Description: Minimum order quantity: 1 unit of 1KIT

Caspase-8 Colorimetric Assay Kit

K113-25
EUR 207

Caspase-6 Colorimetric Assay Kit

K115-25
EUR 202

Caspase-2 Colorimetric Assay Kit

K117-25
EUR 213

Caspase-3 Colorimetric Assay Kit

K106-25
EUR 207

Caspase-5 Colorimetric Assay Kit

K123-25
EUR 213

Caspase-10 Colorimetric Assay Kit

K125-25
EUR 213

Caspase-4 Colorimetric Assay Kit

K127-25
EUR 229

Caspase-1 Colorimetric Assay Kit

K111-25
EUR 207

Caspase-3 Colorimetric Assay Kit

K2008-25 25 assays
EUR 238

Caspase-1 Colorimetric Assay Kit

K2011-25 25 assays
EUR 238

Caspase-8 Colorimetric Assay Kit

K2013-25 25 assays
EUR 238

Caspase-6 Colorimetric Assay Kit

K2015-25 25 assays
EUR 238

Caspase-2 Colorimetric Assay Kit

K2017-25 25 assays
EUR 251

Caspase-5 Colorimetric Assay Kit

K2196-25 25 assays
EUR 251

Caspase-10 Colorimetric Assay Kit

K2197-25 25 assays
EUR 251

Caspase-4 Colorimetric Assay Kit

K2199-25 25 assays
EUR 266

Caspase-9 Fluorometric Assay Kit

K118-25
EUR 207

Caspase-9 Fluorometric Assay Kit

K2018-25 25 assays
EUR 238

CASPASE3 DEVDR110 ASSAY (25 ASSAYS)

30008-1 1KIT
EUR 204
Description: Minimum order quantity: 1 unit of 1KIT

Caspase Colorimetric Substrate Set II

K134-9-25
EUR 659

Caspase 9 Assay Kit

20-abx299021
  • EUR 1302.00
  • EUR 582.00
  • 100 tests
  • 25 tests

Caspase 9 Assay Kit

20-abx299035
  • EUR 1302.00
  • EUR 582.00
  • 100 tests
  • 25 tests

Caspase-3 DEVD-R110 Fluorometric and Colorimetric Assay Kit (100 assays)

30008-2 1KIT
EUR 383
Description: Minimum order quantity: 1 unit of 1KIT

Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay Kit (100 assays)

30011-2 1KIT
EUR 383
Description: Minimum order quantity: 1 unit of 1KIT

Caspase Colorimetric Substrate Set II Plus

K138-9-25
EUR 833

Caspase 3 Colorimetric Assay Kit

55R-1270 25 assays
EUR 277
Description: Assay Kit for detection of Capase 3 activity in the research laboratory

Caspase 1 Colorimetric Assay Kit

55R-1273 25 assays
EUR 277
Description: Assay Kit for detection of Capase 1 activity in the research laboratory

Caspase 8 Colorimetric Assay Kit

55R-1275 25 assays
EUR 277
Description: Assay Kit for detection of Capase 8 activity in the research laboratory

Caspase 6 Colorimetric Assay Kit

55R-1277 25 assays
EUR 284
Description: Assay Kit for detection of Capase 6 activity in the research laboratory

Caspase 2 Colorimetric Assay Kit

55R-1279 25 assays
EUR 303
Description: Assay Kit for detection of Capase 2 activity in the research laboratory

Caspase 5 Colorimetric Assay Kit

55R-1283 25 assays
EUR 303
Description: Assay Kit for detection of Capase 5 activity in the research laboratory

Caspase 10 Colorimetric Assay Kit

55R-1285 25 assays
EUR 303
Description: Assay Kit for detection of Capase 10 activity in the research laboratory

Caspase 4 Colorimetric Assay Kit

55R-1287 25 assays
EUR 312
Description: Assay Kit for detection of Capase 4 activity in the research laboratory

Caspase-8 Colorimetric Assay Kit

K113-100
EUR 479

Caspase-8 Colorimetric Assay Kit

K113-200
EUR 620

Caspase-8 Colorimetric Assay Kit

K113-400
EUR 958

Caspase-6 Colorimetric Assay Kit

K115-100
EUR 452

Caspase-6 Colorimetric Assay Kit

K115-200
EUR 615

Caspase-6 Colorimetric Assay Kit

K115-400
EUR 958

Caspase-2 Colorimetric Assay Kit

K117-100
EUR 468

Caspase-2 Colorimetric Assay Kit

K117-200
EUR 615

Caspase-2 Colorimetric Assay Kit

K117-400
EUR 958

Caspase-3 Colorimetric Assay Kit

K106-100
EUR 479

Caspase-3 Colorimetric Assay Kit

K106-200
EUR 631

Caspase-3 Colorimetric Assay Kit

K106-400
EUR 958

Caspase-5 Colorimetric Assay Kit

K123-100
EUR 468

Caspase-5 Colorimetric Assay Kit

K123-200
EUR 615

Caspase-5 Colorimetric Assay Kit

K123-400
EUR 958

Caspase-10 Colorimetric Assay Kit

K125-100
EUR 463

Caspase-10 Colorimetric Assay Kit

K125-200
EUR 615

Caspase-10 Colorimetric Assay Kit

K125-400
EUR 958

Caspase-4 Colorimetric Assay Kit

K127-100
EUR 468

Caspase-4 Colorimetric Assay Kit

K127-200
EUR 615

Caspase-4 Colorimetric Assay Kit

K127-400
EUR 958

Caspase-1 Colorimetric Assay Kit

K111-100
EUR 479

Caspase-1 Colorimetric Assay Kit

K111-200
EUR 620

Caspase-1 Colorimetric Assay Kit

K111-400
EUR 958

Caspase-3 Colorimetric Assay Kit

K2008-100 100 assays
EUR 474

Caspase-3 Colorimetric Assay Kit

K2008-200 200 assays
EUR 696

Caspase-3 Colorimetric Assay Kit

K2008-400 400 assays
EUR 1086

Caspase-1 Colorimetric Assay Kit

K2011-100 100 assays
EUR 474

Caspase-1 Colorimetric Assay Kit

K2011-200 200 assays
EUR 696

Caspase-1 Colorimetric Assay Kit

K2011-400 400 assays
EUR 1086

Caspase-8 Colorimetric Assay Kit

K2013-100 100 assays
EUR 474

Caspase-8 Colorimetric Assay Kit

K2013-200 200 assays
EUR 696

Caspase-8 Colorimetric Assay Kit

K2013-400 400 assays
EUR 1086

Caspase-6 Colorimetric Assay Kit

K2015-100 100 assays
EUR 487

Caspase-6 Colorimetric Assay Kit

K2015-200 200 assays
EUR 696

Caspase-6 Colorimetric Assay Kit

K2015-400 400 assays
EUR 1101

Caspase-2 Colorimetric Assay Kit

K2017-100 100 assays
EUR 529

Caspase-2 Colorimetric Assay Kit

K2017-200 200 assays
EUR 725

Caspase-2 Colorimetric Assay Kit

K2017-400 400 assays
EUR 1156

Caspase-5 Colorimetric Assay Kit

K2196-100 100 assays
EUR 529

Caspase-5 Colorimetric Assay Kit

K2196-200 200 assays
EUR 725

Caspase-5 Colorimetric Assay Kit

K2196-400 400 assays
EUR 1156

Caspase-10 Colorimetric Assay Kit

K2197-100 100 assays
EUR 529

Caspase-10 Colorimetric Assay Kit

K2197-200 200 assays
EUR 738

Caspase-10 Colorimetric Assay Kit

K2197-400 400 assays
EUR 1114

Caspase-4 Colorimetric Assay Kit

K2199-100 100 assays
EUR 557

Caspase-4 Colorimetric Assay Kit

K2199-200 200 assays
EUR 738

Caspase-4 Colorimetric Assay Kit

K2199-400 400 assays
EUR 1114

Caspase 9 Fluorometric Assay Kit

55R-1280 25 assays
EUR 277
Description: Assay Kit for detection of Capase 9 activity in the research laboratory

Caspase-9 Fluorometric Assay Kit

K118-100
EUR 468

Caspase-9 Fluorometric Assay Kit

K118-200
EUR 615

Caspase-9 Fluorometric Assay Kit

K118-400
EUR 958

Caspase 9 Assay Kit, green

KF17206 25 Tests
EUR 304

Caspase 9 Assay Kit, red

KF17302 25 Tests
EUR 304

Caspase-9 Fluorometric Assay Kit

K2018-100 100 assays
EUR 502

Caspase-9 Fluorometric Assay Kit

K2018-200 200 assays
EUR 669

Caspase-9 Fluorometric Assay Kit

K2018-400 400 assays
EUR 1114

Caspase-6 Fluorometric Assay Kit

K114-25
EUR 202

Caspase-2 Fluorometric Assay Kit

K116-25
EUR 202

Caspase-3 Fluorometric Assay Kit

K105-25
EUR 207

Caspase-10 Fluorometric Assay Kit

K124-25
EUR 202

Caspase-4 Fluorometric Assay Kit

K126-25
EUR 213

Caspase-12 Fluorometric Assay Kit

K139-25
EUR 256

Caspase-5 Fluorometric Assay Kit

K122-25
EUR 202

Caspase-1 Fluorometric Assay Kit

K110-25
EUR 207

Caspase-8 Fluorometric Assay Kit

K112-25
EUR 207

Caspase-3 Fluorometric Assay Kit

K2007-25 25 assays
EUR 238

Caspase-1 Fluorometric Assay Kit

K2010-25 25 assays
EUR 238

Caspase-8 Fluorometric Assay Kit

K2012-25 25 assays
EUR 238

Caspase-6 Fluorometric Assay Kit

K2014-25 25 assays
EUR 238

Caspase-2 Fluorometric Assay Kit

K2016-25 25 assays
EUR 238

Caspase-12 Fluorometric Assay Kit

K2150-25 25 assays
EUR 251

Caspase-5 Fluorometric Assay Kit

K2195-25 25 assays
EUR 238

Caspase-4 Fluorometric Assay Kit

K2198-25 25 assays
EUR 251

human MMP-9 activity assay kit 96-assays

QZBmmp9H 1 plate 96 assays
EUR 501

mouse MMP-9 activity assay kit 96-assays

QZBmmp9M 1 plate 96 assays
EUR 501

hydroxyproline assay kit 96-assays

QZBhypro1 1 plate 96 assays
EUR 323

Caspase-9, human recombinant

1089-25
EUR 283

Caspase Colorimetric Substrate Set

K132-7-25
EUR 544

Fluorescein Active Caspase-9 Staining Kit

K2051-25 25 assays
EUR 238

Red Active Caspase-9 Staining Kit

K2055-25 25 assays
EUR 238

Caspase 9 Colorimetric Cell-Based ELISA Kit

EKC1089 100ul
EUR 572

Caspase-9, human recombinant protein

E1009-25 25 units
EUR 308
Description: Caspase-9 belongs to the caspase-family of cysteine proteases. Caspase-9 also exists in cells as an inactive proenzyme like other caspases. Procaspase-9 is processed at aspartate residues to form active caspase-9 during the initiation of apoptosis.

Acetylcholinesterase Activity Assay Kit - 100 Assays

AR4001-unit unit