Technical Service Walkthrough
Antibody / antiserum / protein
Specify the name of the product:
Nature of the problem and previous experience
1. What is the specific problem you are experiencing?
2. Did the same vial of product work in the past? What were the results?
3. Did other lots of this product work in the past? Which lots? What were the results?
4. How have you reconstituted the lyophilized product (diluent, volume)?
Samples and controls
1. Species (animal) of the sample
2. Type of sample (cell lysate, tissue, purified protein, …) and lysis buffer used
3. How much sample was loaded on the gel?
4. What positive and negative controls were used? What were the results?
Performing the experiment
1. Was a blocking performed? If yes, describe it (blocking solution, time, temperature).
2. At what temperature has the primary antibody been stored? Has it ever been frozen?
3. At what dilution was the primary antibody used and what was the diluent?
4. What were the incubation conditions (time, temperature) for the primary antibody?
5. What was the secondary antibody used (company, HRP or AP conjugated, against which species, etc)?
6. At what dilution was the secondary antibody used and what was the diluent?
7. What were the incubation conditions (time, temperature) for the secondary antibody?
8. Has a secondary control been performed successfully?
9. Can you verify that all molecular weight markers are transferred to the membrane?
10. What detection system was used and what was the exposure time?
11. What type of membrane was used? (PVDF, nitrocellulose)
12. What percentage of gel was used? Was this appropriate for the size of the protein?
13. Would you have pictures to image the situation?
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