Anti-PTBP1 抗体 (ab5642)

Thank you for the details that you have provided. At this point, I would like to make the following suggestions. Use ab5642 at a concentration of 1 µg/ml and incubate overnight at 4C. Do not re-use the primary antibody. Ensure that your secondary antibody is working properly, and you may need to increase the concentration of the secondary antibody as well as the incubation period. Do not overwash the membrane. Also, I suggest running a positive control - Jurkat lysate is recommended as positive control for ab5642.

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab5642. A secondary antibody that is compatible for use with this primary is ab6741 - Rabbit polyclonal to Goat IgG H&L (HRP). I would like to help you out and if you would kindly answer the questions below, the answers will enable us to investigate this matter as quickly as possible. Also, please include the batch number that you received (it is located on the vial) and the Abcam order number or purchase order number that was used. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? 6. What detection method are you using? 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify.