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For IHC, we recommend combining the peptide and the primary antibody at a 10:1 ratio (µg : µg) in the antibody diluent. Incubate the peptide/antibody mixture for a minimum of 30 minutes prior to adding the entire volume to the slide. The recommended antibody dilution can be found on the product's web page.

 

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Yes. In our protocol, we recommend a probe-based sonicator because it is less expensive and widely used in many labs. However, you can use a Covaris water bath sonicator. You will still need to optimize the sonication program based on your DNA image, since you want to avoid over-sonicating when performing sonication-ChIP as this can damage chromatin quality/integrity. When looking at sonication results, chromatin DNA fragments should appear as a smear typically ranging between 200 to 1000 bp in size. We recommend you use the condition that requires the minimal sonication cycles, as long as the majority of DNA is less than 1000bp.

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We observe multiple bands at or around 33-35 kDa in cells with high STING protein expression, such as THP-1 and HDLM-2. Regrettably, we are not certain about what the lower molecular weight band represents in the THP-1 extract. However, customers may be interested in the following possibilities:

1) Please see Wang, P. et al. (2018) Nucleic Acids Res. 46, 4054-4071 (PMID: 29547894; https://pubmed.ncbi.nlm.nih.gov/29547894/). The two bands in THP-1 extract may represent the alpha and beta isoforms of STING.

2) Please see Rodríguez-García, E. et al. (2018) Immunohorizons 2, 363-376 (PMID: 31026807; https://pubmed.ncbi.nlm.nih.gov/31026807/). STING isoforms 2 and 3 do not include #13647’s epitope. However, STING isoform 1 may be detected by #13647.

3) Given that the lower band is also observed in 293T cells transfected with a construct expressing human STING protein (see image on the product webpage; https://www.cellsignal.com//products/primary-antibodies/sting-d2p…

Hypoxia Signaling

Learn here about the HIF-1 signaling pathway and its impact on oxygen homeostasis and cellular adaptation. Click here.

PTMScan® Tyrosine Kinase

Information and case study data for the PTMScan Direct Tyrosine Kinases Service from Cell Signaling Technology.

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The Caspase-4 Antibody #4450 recognizes an epitope that is present in both the full-length protein and the p20 subunit. Therefore, this antibody is predicted to detect both targets. However, we have not confirmed the reactivity of the Caspase-4 Antibody #4450 with the p20 subunit at endogenous levels. The exact protein load of recombinant, active caspase-4 shown in the western image on the product datasheet for the Caspase-4 Antibody #4450 has not been recorded. However, we typically load 1-5 ng of recombinant protein for such experiments.

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We have performed antigen retrieval comparisons with a number of products.  We consistently see the strongest signals with high pH retrieval buffers.  We have not seen the staining pattern influenced by the choice of retrieval protocol; however, the signal intensity can be affected.  Antibodies that yield an acceptable signal with low pH retrieval (citrate buffer) will generally work well with high pH retrieval (EDTA or Tris-EDTA), but the optimal concentration may not be the same.  Antibodies that require the more robust high pH retrieval are unlikely to work as well with low pH retrieval. It is generally our recommendation to use EDTA retrieval when assembling antibodies in a multiplex panel.
 

Webinar - Antibody Validation Roundtable: Specificity + Sensitivity + Reproducibility = Validation

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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. 

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We have performed CUT&RUN on dozens of cultured adherent and suspension cell lines, both human and mouse. So far we have not noticed any significant difference in performance between these cell lines.

Webinar - Immunolabeling for Neurodegenerative Diseases

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We have performed several freeze/thaw experiments and have found that the reagents of the XTT Cell Viability Kit #9095 are very stable after 5 freeze/thaw cycles. It is also fine to aliquot the reagents; this will not negatively impact the activity of the kit. If you choose to aliquot the reagents, make sure they are completely thawed and there is no observable precipitate. If you do notice precipitate in the bottle, the bottle may be warmed in a water bath until it comes into solution. We do not recommend pre-mixing the XTT reagent and Electron Coupling Solution for long term storage.

Antibody Validation Roundtable: Specificity + Sensitivity + Reproducibility = Validation

Webinar - The Potential of Proteomics: A Clear Path to Discovery - Part II