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SignalStar™ Multiplex IHC Troubleshooting Guide

Antibody Validation for SignalStar™ Multiplex IHC

SignalStar™ Spatial Profiling

SignalStar™ Spatial Profiling is a revolutionary new tool for spatial biology research with fully customizable and highly-validated antibody IHC panels.

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Our optimized tagmentation conditions for the CUT&Tag assay work well across multiple cell lines, both adherent and suspension. We recommend using 2 μL of CUT&Tag pAG-Tn5 (Loaded) #79561 in each 50 μL of tagmentation reaction. Using a larger volume of pAG-Tn5 in each reaction doesn't increase the number of reads, peak number, or signal-to-noise ratio, according to in our in-house tests. In fact, pAG-Tn5 activity is more critical to the success of the CUT&Tag assay than the amount of pAG-Tn5 used.

Please make sure to store the pAG-Tn5 enzyme properly at -20℃ and do not use expired pAG-Tn5, as activity does decline over time. The shelf life of CUT&Tag pAG-Tn5 (Loaded) #79561 is six months. Additionally, we have optimized the salt concentration in our tagmentation buffer to ensure efficient target-specific tagmentation and to limit non-specific tagmentation at open chromatin regions.

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When developing the Malachite Green Phosphate Detection Kit #12776, we successfully used the Millipore Centrifuge filters AMICON ULTRA 0.5mL, 10,000 NMWL, product # UFC501096.

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A CUT&RUN assay allows signal normalization among samples or among experiments by adding spike-in DNA in the stop buffer. Since you add the same amount of spike-in DNA in the assay, the signal from spike-in DNA should be the same among all samples or experiments. Any variation of the spike-in DNA signals is due to technical errors that you can exclude by using the normalization strategy. For example, if you accidentally spill one of your samples during the experiment, or if you see lower signal with fewer starting cells, you know that the weaker signal does not represent the real binding strength of the protein on DNA in these scenarios. The sample normalization strategy can help to represent the real binding enrichment of a target protein on genome. Variation of signal strength introduced during DNA purification, library prep, and NGS can be normalized. That said, the spike-in DNA is totally optional. If you are not quantitively comparing a dataset to any other datasets, you do not need to use the spi…

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Unfortunately, we are not aware if any of our amyloid-beta antibodies will detect the alpha-oligomer of the amyloid-beta protein.

Protect Your Drug Discovery Pipeline

To keep your projects moving forward, you need to count on reagents that will work as expected and be available throughout the lifetime of your study.

The CST Hallmarks of Validation

Know what to look for and dig deeper into the validation data for your antibody to avoid the frustration of ambiguous or misleading results.

PhosphoScan® Phosphorylation Proteomics

PhosphoScan® Phosphorylation Proteomics

How to Identify Senescence in Cells and Tissue

Reliably identify senescence signaling in your samples using biomarkers with a 3-stage approach outlined in this video. No single assay is sufficient, but screening, co-staining, and verification with multiple biomarkers can improve your confidence.

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PI4 Kinase Antibody #4902 detects endogenous levels of total PI4 Kinase isoform 230. In humans, PI4K230, PI4K92, and PI4K55 are four PI4 kinase isoforms that have been characterized and classified according to their molecular weights of 230, 92, and 55 kDa.  In the literature, alpha refers to the 230 kDa size band (UniProt ID: P42356) and beta refers to the 92 kDa size band (UniProt ID:Q9UBF8). Type 2 alpha (UniProt ID: Q9BTU6 ) and type 2 beta (UniProt ID: Q8TCG2) refer to the 55 kDa band.

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Bmi1 undergoes numerous post-translational modifications and can be heavily phosphorylated, as described on the PhosphoSite.org protein page (https://www.phosphosite.org/proteinAction.action?id=1286622&showAllSites=true). Therefore, we believe the upper band represents phosphorylated Bmi1.

Multiple papers have also described Bmi1 migrating as a doublet by western blot, as shown in the examples below.

PTMScan® Glycosylation

PTMScan® Glycosylation

Caspase Cleavage Substrate Proteomics

Caspase Cleavage Substrate Proteomics

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We recommend using our Cell Lysis Buffer (10X) #9803 for immunoprecipitation (IP) or co-IP experiments. While both Cell Lysis Buffer(1X) #9803 and RIPA Buffer (1X) #9806 are suitable for generating whole cell extracts for western blotting, RIPA buffer contains the ionic detergent sodium deoxycholate, which has been known to denature kinases and prevent protein-protein interactions, making it less suitable for IPs.
 

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Due to the expected and typically low yield of CUT&Tag DNA, we recommend using all 30 µL of the CUT&Tag DNA sample (from Section VI of the CUT&Tag protocol #77552) for library amplification. While CUT&Tag DNA libraries generated for histone modifications typically show some signal on the Agilent Bioanalyzer or TapeStation systems, libraries generated for non-histone proteins such as transcription factors and cofactors often have very weak or even no visible signal on Bioanalyzer or TapeStation systems, but they still generate NGS results with high-mapping rates, high numbers of identified binding peaks, and decent signal-to-noise ratios across the whole genome. For CUT&Tag libraries where the Bioanalyzer or TapeStation system is unable to identify the average size of the library, we suggest using a size o…

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The NDRG1 target has 3 isoforms produced by alternative splicing (see Q92597). Isoform 2 and isoform 3 are missing amino acids 1-66 and amino acids 1-81, respectively. Depending on the species and cell line/tissue, each of the isoforms could be expressed at varying levels. Different treatments can also lead to varying amounts of isoform expression.

The respective antigens for NDRG1 (D8G9) XP® Rabbit mAb #9485, NDRG1 (D10F8) Rabbit mAb #9395, and NDRG1 Antibody #5196 correspond to residues in the carboxy terminus of human NDRG1 protein. Therefore, #9485, #9395, and #5196 can detect all 3 isoforms, leading to the observation of multiple bands. The antigen for NDRG1 (D6C2) Rabbit mAb #9408, on the other hand, corresponds to residues near the amino terminus and this antibody can only detect isoform 1. 
 

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Reagents that contain casein, such as milk, may result in a reduced signal or lack of signal with some antibodies in IHC (most notably with phospho-specific antibodies). Therefore, casein in the blocking solution for IHC may be problematic. Given this, we routinely block in 1X TBST+5% Normal Goat Serum #5425 (NGS) or Animal-Free Blocking Solution (5X) #15019 when performing IHC experiments.

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The antigen for our Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody #4228 shares 100% sequence homology with human. However, we have tested many different human samples in-house and have not been able to obtain signal with this antibody.

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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.

UbiScan® - Ubiquitination Proteomics

UbiScan® - Ubiquitination Proteomics

Deciphering Immuno-Oncology: Targeting Cellular Mechanisms of the Tumor Immune Response

Key signaling pathways modulate immune cell activation and tumor checkpoint evasion. Improved understanding of tumor microenvironment crosstalk and mechanisms of response/reistance will be key for next-generation cancer immunotherapies.

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It is difficult to predict when the Fc receptor blocking step is necessary, due to the variable interaction of host species with different antibody isotypes and the interference caused by fluorescent dyes directly conjugated to the antibody Fc domain. The presence of these dyes is often sufficient to disrupt Fc receptor binding. If you are unable to conduct an experiment to assess nonspecific binding, we recommend using the CD16/CD32 (2.4G2) Rat mAb #88280 to prevent any unintended antibody binding to the Fc receptor.

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Our Cell Lysis Buffer #9803 will lyse whole cell membranes. Please keep in mind that sonication is crucial to maximize protein recovery when using Cell Lysis Buffer #9803 as sonication will ensure ample nuclear rupture and DNA shearing. Please refer to our recommended lysing protocol for Cell Lysis Buffer #9803 on the product webpage: http://www.cellsignal.com/products/9803.html. If you are looking to separate out fractions (cytoplasmic, membrane/organelle, nuclear/cytoskeletal) when lysing your cultured cells, feel free to consider our Cell Fractionation Kit (#9038).

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This decision was based on our analysis of hundreds of CUT&RUN samples. We found that a sequencing depth lower than 3 million generated too few peaks and that the number of peaks increased with deeper sequencing. If desired, one can increase the sequencing depth to 10 million reads per sample. The duplication rate of sequencing reads significantly increases if the sequencing depth is >15 million reads per sample.

How CUT&RUN Profiles Chromatin

This animated explainer shows how the CUT&RUN method works for mapping protein-DNA interactions in native chromatin, while using fewer cells and saving you time.

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In section C, part 8 of the Senescence β-Galactosidase Staining Kit #9860 protocol, you may overlay the plates with 70% glycerol directly after removing the staining solution. It is not necessary to wash off the staining solution prior to overlaying with glycerol.

SignalScan™ Peptide Mix (SARS-CoV-2)

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The Anti-rabbit IgG, HRP-linked Antibody #7074 and the Anti-mouse IgG, HRP-linked Antibody #7076 have been cross-adsorbed against human IgG. The Anti-rat IgG, HRP-linked Antibody #7077 has been cross-adsorbed against mouse serum.