For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
1. Order details: • Batch number: SA1217-AB5986 • Abcam order or Purchase order number: B09501149 • Antibody storage conditions (temperature/reconstitution etc) ????10????,???4???;????????? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). no band 3. On what material are you testing the antibody in WB? • Species: cell line • Cell extract or Nuclear extract: Cell extract • Purified protein or Recombinant protein: Recombinant protein 3. The lysate • How much protein was loaded: 30?g • What lysis buffer was used: RIPA • What protease inhibitors were used: co cktail(Rocho) • What loading buffer was used: 2x sample buffer • Did you heat the samples: temperature and time: Yes,95? for 5 min. 4. Electrophoresis/Gel conditions/ Transfer conditions • Reducing or non reducing gel: I don`t understand what you mean. • Gel percentage : stacking: 5?; separating:12.5? • Transfer conditions: 70V for 40min 5. Blocking conditions • Buffer: 5?MILK(in TTBS) • Blocking agent: milk, BSA, serum, what percentage: 5?MILK(in TTBS) • Incubation time: 1hr • Incubation temperature: Room Temperature 6. Primary Antibody • Specification (in which species was it raised against): on ice • At what dilution(s) have you tested this antibody: 1:1000 • What dilution buffer was used: 5?MILK(in TTBS) • Incubation time: O/N • Incubation temperature: 4? • What washing steps were done: TTBS 15min/?X4 7. Secondary Antibody • Specification (in which species was it raised against)? on ice • At what dilution(s) have you tested this antibody: 1:1000 • Incubation time 1hr • Wash steps: TTBS 15min/?X4 • Do you know whether the problems you are experiencing come from the secondary? no 8. Detection method ECl, ECl+, other detection method: ECl 9. Background bands • Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): No, I haven`t. • Is the blocking step sufficient? Yes. • Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes • At what size are the bands migrating? Could they be degradation products of your target? Between17-24 kd • Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) • ? 10min ? 1hr 11. Did you apply positive and negative controls along with the samples? Please specify. No 10. Optimization attempts • How many times have you tried the Western? 1 • Do you obtain the same results every time e.g. are background bands always in the same place? • What steps have you altered? Please send this questionnaire by e-mail by cutting and pasting the text in your e-mail (no attachements other than images) to email@example.com.
Asked on Mar 08 2006
Thank you for the details that you have provided and I'm sorry to hear that you are experiencing difficulty with ab5986. At this point I would like to make the following suggestions to help improve results as you are not seeing any bands. This antibody was characterized for use in Western blotting with A431 lysate and I strongly suggest using this as a positive control. I also suggest increasing the amount of primary that you are using to a dilution of 1:500 and incubating overnight at 4C. Also, you want to ensure that the protein transferred properly to the membrane (check this with Ponceau S) and check that your secondary is working properly. Is the secondary working with other primaries? You may also need to increase the incubation period and concentration of your secondary. I hope this helps. Please contact us again if you need additional assistance.
Answered on Mar 09 2006