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Applications FAQs

Creative Biolabs answers common applications FAQs (Frequently asked questions) to accelerate your project development.

  1. What applications can your antibodies be used for?

    2. We have thousands of antibodies that can be used in various applications, such as WB, IF, FC, IP, IHC, ELISA, CHIP, ELISpot and so on.

  2. How can I choose the correct antibody for my experiment? Do you provide antibodies with multiple applications?

    3. The choice of a suitable antibody product depends on different requirements including the experimental purpose, the species of samples, the host species of antibodies and the labeling of antibodies. Our various antibodies can meet your application requirements and detailed information you required are listed on the website or product data sheet. If we did not test an application that you are looking for, we will offer you a trial size sample to evaluate the antibody before purchasing full size.

  3. What is the recommended concentration to use your antibody in western blot analysis?

    4. Normally, it is recommended to use our antibody in WB with a dilution range of 1:500-1:2000. For more detailed information, please view our Product page.

  4. What is the recommended concentration to use the antibody in ELISA analysis?

    5. Generally, the suitable dilution for ELISA should be determined by the actual experiment and depends on customer's assay platform and system. You may contact us and our professional scientists are pleased to provide you an optimal solution.

  5. Can you please offer me the protocol for WB, FC, IF, IP, ChIP, ELISA or IHC?

    6. To obtain the protocol, please see the Protocols & Troubleshooting & Guide page, or we also can send you the protocols via e-mail.

  6. Why does the actual Western blot band sometimes differ in size compared to predicted size?

    7. Western blotting is a technique to separates proteins mainly depending on the different protein sizes. Thus, the actual band size often differs from a predicted size due to the following factors:

    • Splice variants. One gene can generate proteins of different sizes due to alternative splicing.
    • Post-translational modifications. Such as glycosylations and phosphorylations can increase the molecular weight of the target proteins.
    • Post-translational cleavage. The cleavage of pro-proteins to generate the active protein leads to a change of the molecular weight.
    • Dimers and multimers.
    • Hydrophobic proteins. Transmembrane proteins are typical hydrophobic proteins that have difficulties in migrating through the gel resulting in different multi-bands patterns.
  7. Why are there no bands in western blot (WB)?

    8. Some reasons are present as follows:

    • Lacking of antibody. You may reduce antibody dilution. Besides, antibody may have lost activity, and a dot blot assay can confirm the factor.
    • Lacking of protein. We recommend increasing the amount of total protein loaded on the gel.
    • Poor transfer. We recommend using wet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer to ensure good contact between PVDF membrane and gel. In addition, some proteins with high molecular weight, we recommend you extend transfer time. By contrast, for some proteins with small molecular weight (lower than 10 kDa), you should reduce transfer voltage or time.
    • Incorrect secondary antibody used. Confirm your antibody matches host species and choose the right secondary antibody.
    • Sodium azide contamination. Sodium azide can quench HRP signal so you should make sure buffers without it.
    • Insufficient incubation time for primary antibody. Extend incubation time to overnight at 4℃.
  8. Why is the signal weak in western blot (WB)?

    9. Possible reasons include the following:

    • Low protein-antibody binding. We recommend minimize the number of washes.
    • Insufficient antibody.
    • Inactive conjugation.
    • Weak/Old ECL. We suggest purchasing new ECL reagents.
    • Non-fat dry milk may mask some antigen. Decrease percentage of blocking milk or substitute with 3% BSA.
  9. Which applications are the antibodies validated for?

    10. Our antibodies are validated for at least one or more applications such as IHC, WB and ICC. All the information about the applications of an antibody has been listed in the antibody product page.

If a certain antibody is not yet recommended in you need application testing or species, you are welcome to contact us for a sample.

If you still have other questions, please feel free to contact us.

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