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We recommend using 10 mg of protein per immunoaffinity purification (IAP) sample for our standard PTMScan kits. For the PTMScan HS product line, 1 mg of starting material is optimal. The minimum sample amount is dependent upon the cell line/tissue and the endogenous levels of post-translational modifications (PTMs). If less protein is used, fewer identifications could be expected.

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Any tissue shown to be positive for each target via chromogenic IHC can serve as a positive control tissue for a SignalStar™ assay. Each target will therefore require a positive control, so each assay may require multiple controls depending on the targets chosen. For optimal comparison, the sections should be as close to serial as possible.

Immunofluorescence Protocol for Cell-based Assays Requiring Methanol Fixation

Immunofluorescence Protocol with Methanol Fixation (IF Methanol-fixed): easy to follow directions describing the step by step experimental procedure.

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While we recommend 1mg of tissue per assay in most cases, we have had some success with as little as 0.5 mg of tissue per assay. Easier tissue types, such as liver, or easier target types, such as histone modifications, require less tissue. Difficult tissue types like heart or difficult target types like some transcription factors or cofactors will likely require greater amounts of tissue, such as 2.5mg or 5mg.

Mechanisms of Cell Death: Necrosis & Necroptosis

Activated by pro-inflammatory signaling, ischemic injury or viral infection, necroptosis is a programmed regulated form of necrosis requiring RIP3 and MLKL

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The typical DNA yields from ChIP experiments range from 0.2-2 ng/µl when targeting transcription factors or cofactors and from 2-20 ng/µl when targeting histone modifications. If the concentration is within this range, then you should be able to continue with ChIP-seq. Our DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 can generate a library with 0.5ng ChIP-DNA.

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The amount of CUT&RUN DNA required for NG-seq analysis depends on the sensitivity of the DNA library prep kit. The typical yield for CUT&RUN DNA is 0.6 to 6 ng per reaction. Our SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795 will work with starting DNA as low as 0.5 ng. You can also increase the number of PCR cycles up to 20 to increase amplification of the library if the starting amount is too low.

Enzyme-linked Immunosorbent Assay (ELISA) Components

In addition to the samples to be analyzed, other required materials in order to run a complete ELISA experiment include microplates, reagents, buffers, antibodies, and a plate reader.

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We suggest a sequencing depth of 3-5 million reads per sample. Because of the very low background signal generated in CUT&RUN, this sequencing depth is usually sufficient for histone modifications and transcription factors. We have found that less than 3 million does not work and greater than 15 million greatly increases the number of duplicate reads.

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Yes, the flow through from a PTMScan experiment can be saved and stored at -80C for future enrichments. We have performed 5 different PTM enrichments sequentially from one sample. The limitation would be the choice of the initial protease used for the digest. For example, a UbiScan PTMScan requires peptides digested with trypsin and the flow through peptides may not be ideal to capture peptides that require an arginine (R) residue in the phospho-motif as in the AKT or AMPK substrate PTMScan kits.

Metabolism

High-quality antibodies & assays supporting the interrogation of metabolic signaling pathways. Those required for energy, autophagy & biochemical reactions.

California Transparency in Supply Chains Act of 2010

The California Supply Chains Act requires CST to disclose its current practices to combat slavery and human trafficking in five specific areas.

Tips for Flow Cytometry Success

To perform intracellular flow cytometry, fixation and permeabilization of cells is required to allow for antibody penetration and subsequent binding to targets without disrupting cellular morphology.

SimpleChIP® - ChIP Assay Kits

Our ChIP assay kits avoid the problems of traditional sonication protocols, which require subjecting chromatin to harsh, denaturing conditions. Learn more.

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In our experience, digesting 4x10^6 cells yields 10-20 ug of chromatin, which is why this is mentioned in our ChIP FAQ. However, as stated in the protocols for our SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 and SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, approximately 5ug - 10ug chromatin is required for ChIP depending on your target. In our experience, for histone (PTM) ChIP, as low as 1-2 ug of chromatin DNA is required per IP. For transcription factor ChIP, a minimum of 5-8 ug chromatin DNA is required per IP. For co-factor ChIP, a minimum of 10 ug chromatin is necessary per IP. This is why all of our validated antibodies have optimal dilutions based on 10ug per IP.

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There are several differences between ChIP and CUT&RUN. CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation, making it a much faster and more efficient method for enriching protein-DNA interactions and identifying target genes. Instead of fragmenting all of the cellular chromatin as done in ChIP, CUT&RUN utilizes an antibody-targeted digestion of chromatin, resulting in much lower background signal than seen in the ChIP assay. As a result, CUT&RUN requires only 1/10th the sequencing depth that is required for ChIP-seq assays.

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As long as the image is not being used for commercial use, we only require for you to acknowledge us with the text "Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com)". This statement should be clearly represented with the image in all media.

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The FastScan kits differ from the PathScan kits in that (1) they come with a positive control and (2) the assays can be performed much faster. With FastScan, the samples are incubated with a capture antibody conjugated with a proprietary tag and a detection antibody linked to HRP with no need for wash steps. The FastScan kits require the use of a specific lysis buffer, included in the kits, and are not compatible with other buffers. In terms of sensitivity, FastScan and PathScan are fairly equivalent.

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While performing the BrdU cell proliferation assay, we also notice some changes in cell morphology after adding the fixing/denaturing solution. However, in our experience, it does not adversely affect the signal obtained with the kit. It should be noted that we do not recommend any washes after fixation/denaturation. Instead, we recommend waiting until after incubating the detection solution for one hour at room temperature. No centrifugation step is required unless starting with suspension cell lines.

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Our Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile) #9872 (both L and S sizes) is tested for sterility and endotoxin levels. All new lots are subjected to a 2 week sterility test and also tested to ensure an endotoxin level of less than 0.1 EU/ml PBS. We do not do any specific testing to certify the buffer as DNAse/RNase free, therefore we would not recommend using #9872 for experiments requiring RNAse free reagents.

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We recommend using a microwave to bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0 and then to maintain at sub-boiling temperature for 10 minutes. This typically requires heating slides at full power for 2.5 -3 minutes, then adjusting to 20-30% power. After microwave heating, we cool the slides on the bench top for 30 minutes.

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The main difference between our SimpleChIP Enzymatic Chromatin IP Kits (#9002 & #9003) and our SimpleChIP Plus Enzymatic Chromatin IP Kits (#9004 & #9005) is that the SimpleChIP Plus Enzymatic Chromatin IP Kits contain a protocol for fixing and preparing chromatin from tissues and the required additional material to do so (which is the reason for the price difference).

GPCR, Calcium, cAMP

Overview of GPCR, Calcium, cAMP Signaling Resources, interactive pathway diagrams, and other technical resources.

Annual Grants Program

Annual grant application guidelines and project description criteria for funding from CST.

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We have performed retrieval comparisons with a number of antibodies and found, generally, that EDTA tends to result in stronger signals, both specific and potentially non-specific. If antibodies that are optimized with citrate retrieval are used with EDTA, further titration may be required in order to achieve an optimal signal. Conversely, use of citrate in some instances where EDTA is recommended could contribute to a weaker signal.

Tools for Screening and Quantifying Proteins

No matter your research area, we’ve all needed to answer this deceptively easy question: how much of a particular protein is present in my samples?

How to Sonicate Chromatin: Enzymatic ChIP Protocol

SignalStar™ Multiplex Immunohistochemistry Assay for Use on BOND RX Fully Automated Research Stainer by Leica Biosystems

Protein Kinase C Signaling

Study the PKC pathway's role in cellular signaling and regulation. Explore the molecular mechanisms & signaling events governed by this pathway. Click.

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Particulates may occasionally be seen in our Animal-Free Blocking Solution (5X) #15019 due to material becoming caught in the threads of the cap/bottle and drying out over a short period of time. These particulates are of no cause for concern and you may continue to use and dilute the product to the 1X working concentration. You may also heat the product at 37C for 15 minutes to help bring any material back into solution, if required.