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  • The expected molecular weight (MW) listed in the product datasheet is based solely on the size of the target protein’s amino acid sequence. It’s important to remember that many factors can affect the banding pattern of your western blot including: (1) the existence of a splice variant, (2) the quality of the loaded sample, (3) the protein extraction method, and (4) the protein transfer conditions. To achieve accurate results, you may need to systematically adjust the protein extraction method or the protein transfer conditions.

    • If the band’s MW is below the expected MW it could be due to a splice variant with a slightly different MW.
      • Heat the samples at 70°C for 10 min.
      • Increase the transfer time.
    • If the band’s MW is above the expected MW, it could be due to post-translational modifications.
    • In either event, you should use a blocking peptide as a negative control.

  • Some toxins and peptides are soluble in DMSO; you can find this information in the product specification section. For these products, prepare a concentrated stock solution first.

    1. Centrifuge vial (10,000 x g, 5 minutes) before adding solvent.
    2. Dissolve the lyophilized reagent in DMSO. We recommend keeping a concentrated stock solution at 1-10 mM (100-1000X higher than the final working concentration).
    3. Once the peptide is completely dissolved in DMSO, slowly dilute the peptide in pure water (or buffer) to the desired final working concentration.

    Note: We recommend that you maintain a DMSO concentration as low as possible. For cell assays, a final concentration of 0.1–0.5% DMSO (v/v) is considered acceptable. For other experiments, 5% DMSO (v/v) is recommended; adjust this according to your experimental requirements.

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Category: Flow Cytometry Antibodies
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