Anti-S100 beta antibody [SH-B1] (ab11178)

I'm sorry to write back to you quite late. The size of bands on my gel were 118 kd, 80 kd, 70 kd, 50 kd, 32 kd, 25kd, 20 kd, 18kd, 16 kd, 15 kd. I got lots of bands but they were appeared on the gel of 'secondary only' with exactly same patterns. I didn't try other type of samples. I ran three different type of samples; whole testicular cells, interstitial cells in testes, and male germ cells. the order no. is 58484 and lot no, of the antibody is 54995.

ANSWER:

Thank you for your email. It sounds like your secondary antibody is binding non-specifically. When you run a secondary-only control (no primary antibody) you should not see any bands on the membrane. What I suggest is decreasing the concentration of the secondary antibody, and if possible, use a different secondary for comparison. If you have any more questions, please let me know.

Dear Dr. Bagrij,

I sent you mail about the S100-b antibody that didn't work. After the mail I have manipulated almost every condition to make it work. but the antibody still didn't work I'll give you the answer of the questions below you wanted me to answer before. Please think about the problems and if you don't think it'll work, please consider to refund to me. Thanks!

1. Please describe the problem (high background, wrong band size, more bands, no band etc). no band on the proper location and high non-specific bands

2. On what material are you testing the antibody in WB? Species? Domestic cats cell extract/ Nuclear extract? Cell extract Purified protein? Recombinant protein?

3. How much protein did you load? 25 ng, 50 ng, 75 ng, 100ng how did you prepare the lysate for the analysis (protease inhibbitors etc)? I used protease inhibitors Did you heat the samples? Yes, I heated the samples in 120 C heat block for 5 minutes.

4. Primary Antibody Specification (in which species was it raised against)? Mouse anti-bovine At what dilution(s) have you tested this antibody? 1:1000, 1:200 Incubation time, wash step? 1hr at RT, 4 hr at RT, overnight at 4C

5. Secondary Antibody Specification (in which species was it raised against)? Sheep anti-mouse (HRP linked) what dilution(s) have you tested this antibody? 1:5,000, 1:10,000 Incubation time, wash step? 1 hr, three times for 10min Do you know whether the problems you are experiencing come from the secondary? I dont have any problem with the secondary antibody yet

6. What detection method are you using? detected on the film using by ECL solution

7. Background bands Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a No primarycontrol) I did the sample of secondary only (no primary)

Is the blocking step sufficient? (We recommend blocking the memmbrane 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) I performed the blocking step with 5% non-fat dry milk with 0.1 % Tween-20 in TBS and 5% gelatin with 0.1% Tween-20 in TBS as blocking solution and incubated for 1 hr at RT, 2 hr at RT and overnight at 4C.

Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) I did multiple short washes; for 10 minutes, three times

At what size are the bands migrating? Could they be degradationn products of your target? The expected size of samples is 10.5 Kd. Because the size is pretty small, I run the samples in 12.5 % SDS-PAG until dye front hit the little over the middle of lower gel and I also manipulated the transfer time to 30 minutes, which I usually do for 2 hours.

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 Wesern? 10 times

Do you obtain the same results every time e.g. are background bands always in the same place? Yes

What steps have you altered? Incubation time, temperature and concentration for primary and secondary antibody, blocking solution, incubation time, and temperature for blocking, transfer time, used freshly extracted protein, fresh antibody

9. Did you apply positive and negative controls along with the samples? Please specify.

I hope I can hear you ASAP.

ANSWER:

Thank you for your email. My colleague Dr. Bagrij is currently out ill and so I'm replying back to you. Can you tell me at approximately what size are the bands migrating at on your gel? I know that you are not seeing the expected band at 10.5 kDa but I'm curious as to the size of your other bands. Have you tried running any other types of samples for comparison? Also, you mentioned that you ran a secondary antibody control (no primary antibody); what did you see?

I also need to know your order number - the Abcam order number or the purchase order number that was used. Thank you, and I look forward to hearing from you.

I don't have technical problems yet. but I just want to know where this antibody is from. I bought antibody of s100-b from Japanese company which is JIMRO (www.jimro.co.jp). i wonder the antibodies from your company and Jimro are from same resource. Thanks.

ANSWER:

Thank you for your email. We produce some of our antibodies in-house and others are sourced from suppliers whose products we believe will be of interest to our customers. This product falls into the latter category. The identity of our suppliers remains commercially sensitive information. It is not, however, produced by JIMRO. If you have any more questions, please contact us again.