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We do not recommend using multiple unconjugated antibodies from the same species in a flow cytometry experiment because it will not be possible to detect each antibody target independently. If your experiment requires the use of multiple antibodies from the same host species, we suggest using antibodies that are directly conjugated to a fluorophore. If a fluorescent conjugate for your antibody of interest is not available, consider using our custom conjugation services to create a solution that will fit the needs of your experiment.
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Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108, Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4) Rabbit mAb #8828, Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb #18338, and Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAb #9520 are performance tested on extracts prepared from serum-starved cells that have been treated with 10ng/ml TGF-beta3 for 30 minutes. It is possible that longer treatments (e.g., 24 hours) can lead to diminished signals for phospho-Smad2 and phospho-Smad3 because of Smad7 induction.
Failure to include the appropriate serine/threonine phosphatase inhibitors can result in a diminished signal and/or inconsistent results between experiments. We always include sodium pyrophosphate (2.5mM final) and beta-glycerophosphate (1.0mM final) as serine/threonine phosphatase inhibitors in the lysis buffer for detection of the phospho-Smads. Our Phosphatase Inhibit…
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When performing CUT&RUN with downstream qPCR analysis, one can use a normal IgG antibody enriched sample as the negative control and calculate the target-specific antibody enrichment as fold over IgG background. However, the IgG sample typically contains extremely small amounts of DNA and is not a good indicator for how well your target-specific antibody is working in the assay. Therefore, we recommend generating and using an input chromatin DNA sample as well, as described in the protocol of our CUT&RUN Assay Kit #86652. This way, you can express the target-specific antibody DNA enrichment as a percent of the total input DNA and directly determine how well the target-specific antibody is working in the assay.