Cell Signaling Technology

Western Technical Support

NOTE: For the best possible results, Cell Signaling Technology (CST) strongly recommends using our optimized application-specific protocols for each product. These protocols are the result of extensive in-house validation performed at CST and ensure accurate and reproducible results.

To ensure a timely solution, please have the information below ready when you contact us. Technical Support can be reached by toll free phone at 877-678-8324 or 877-678-TECH, fax at 978-867-2400 or e-mail at support@cellsignal.com.

Have you consulted our Western Immunoblotting Troubleshooting Guide before using this technical report? For a listing of recommended cell types and treatments for use as positive controls for our activation state antibodies, visit our Recommended Controls page.

General Questions

  1. Catalog number.
  2. Lot number and reference date printed on tube.
  3. How long have you had the product?
  4. At what temperature was the product stored?
  5. Was the product aliquotted?

Sample Information

  1. Cell type used for the sample lysate: (cell line, primary, whole tissue).
  2. Sample species.
  3. Induction/drug treatment used.
  4. Concentration of the drug used.
  5. What were the time points of induction?
  6. Positive control used.
  7. Lysis buffer used: (SDS Sample Buffer, CST Cell Lysis Buffer, CST Chaps Buffer, CST RIPA Buffer, other).
  8. What specific phosphatase inhibitors, if any, were included in the lysis buffer?
  9. Were the samples sonicated?
  10. Amount of protein lysate loaded per well.
  11. Date of lysate preparation.
  12. Lysate storage temperature: (4°C, -20°C, -80°C, other).
  13. Has this antibody previously worked on these samples?

Protocol Information

  1. Percentage of acrylamide in the gel.
  2. Which membrane was used: (Nitrocellulose, PVDF, Nylon, other).
  3. What is the pore size of the membrane being used for transfer?

Blocking

  1. Components of the blocking buffer.
  2. What was the blocking time?

Primary Antibody Incubation

  1. Primary antibody dilution.
  2. Components of the primary antibody dilution buffer.
  3. Incubation time for the primary antibody.
  4. Washing buffer and time after primary antibody incubation.

Secondary Antibody Incubation

  1. Secondary antibody source.
  2. Secondary antibody dilution.
  3. Components of the secondary antibody dilution buffer.
  4. Incubation time for the secondary antibody.
  5. Washing buffer and time after secondary antibody incubation.

Detection

  1. Which detection system was used: (Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP), other).
  2. Supplier of the detection system.
  3. Length of time for film exposure.
  4. What is the appearance of the exposed and developed film?

Support