Anti-S100 beta antibody (ab14688)

One of our customers works with Tilapia which many of it's genes share high resembles to Zebra fish. The customer would like to get your opinion regarding antibodies suitability and possibly to order some of them as part of the abreview promotion. It is rather difficult to obtain Tilapia tested antibodies but customer submitted reviews on this species might also help understand their reactivity in Zebra fish samples.   The customer wishes to know about chances for success and your willingness to approve abreview promotions for the following antibodies:   Ab85367 Ab88074 Ab868 Ab14688  

ANSWER:

Thank you for contacting us.  Unfortunately, I was unable to locate any amino acid sequences for Tilapia S100, S100 beta, or Dopamine D2 Receptor with which to compare the immunogen sequences for these antibodies.  For this reason, we have no information about whether or not these antibodies might react with Tilapia.  If the customer would still wish to test them, please let me know and I will be happy to set up testing discount codes. 

DESCRIPTION OF THE PROBLEM The Ab should recognize a band at 12kDa, that band is very faint, however, there are approx. 9 other bands including some very distinct bands at ~50 9on 4-20% gel), 80kDa (on 15% gel). I'm not convinced that the band at mw 12 is true band as there are so many others and it is so faint. I was searching for an S100B protein to block the primary when but thought I'd make inquiries first.

SAMPLE human brain homogenate

PRIMARY ANTIBODY 1:500 (no bands at predicted mw at lower dilutions), over night at 4oC, also over night and then 2-3 hours at room temp more

rinse in dH2O, wash 4x in TBST for 5 min each

DETECTION METHOD ECL

ANTIBODY STORAGE CONDITIONS -20oC, no reconstitution (in glycerol)

SAMPLE PREPARATION -Tissue was homogenized in 5mM Tris Hcl with "Complete Tabs" (protease inhib.) -Samples for electrophoresis were denatured/reduced using SDS & B-mercapto EtOH, boiled at 95o for 4 min.

AMOUNT OF PROTEIN LOADED 7 samples ranging from 5-35ug protein (increments of 5ug)

ELECTROPHORESIS/GEL CONDITIONS 15% Tris HCl gel - no bands detected at expected mw 4-20% gradient gel (Tris HCl), ~1 hour @ ~100v

TRANSFER AND BLOCKING CONDITIONS Semi-dry transfer, ~ 1 hour @ 20v Blocked in 5% dry milk in TBST (.05% Tween) for 30 min - 3 hours (have tried range of times)

SECONDARY ANTIBODY 1:5000 anti-rabbit HRP conjugated Ab[ acompetitor] 2 hours room temp

rinse in dH2O, wash 4x in TBST for 5 min each

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10+ HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? gels: 10%, 12%, 15%->no band at expected mw but lots of other bands at varying mw; 4-20% gradient gel gave band ~12kDa Transfer: traditional "wet" overnight transfer at 22v at 4oC, semi-dry transfer (described above) Blocking buffer: tried PBS, PBST, TBS, TBST of varying % dry milk (1, 3, 5, 10%) Ab: tried a range of primary and secondary Ab incubation times and dilutions

ADDITIONAL NOTES Attached is a picture of the most recent attempt on a 4-20% gel. (1 min. film exposure)

ANSWER:

I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionnaire and I have a few comments.

S100 beta is a very small protein. It can be difficult to detect small proteins by using western blotting. Many of our antibodies against small proteins are applied by dot blotting rather than western. The reason why it can difficult is that since smaller polypeptides migrate faster, they may still be heavily coated with SDS when they leave the gel and encounter the membrane, thus reducing the efficiency of protein binding to the membrane. Therefore, for small molecular weight proteins, it is often beneficial to pre-soak the acrylamide gel for 5 to 15 minutes (depending on gel thickness) in transfer buffer without SDS prior to transfer. This will help to reduce the SDS concentration and enhance binding of low molecular weight proteins to the membrane.

Can you please tell me some details of the membrane that you have been using and whether you feel it can capture small protein molecules or whether it is possible that they are not being transferred efficiently.

However, despite a potential inefficient transfer I appreciate that there are abherrant bands that should not be present. I would appreciate it if you could please send me your western images again as unfortunately they did not make it through our system. I look forward to hearing from you and appreciate your patience in this matter.