Anti-PRPF8 antibody (ab79237)

LOT NUMBER GR43386-1 ORDER NUMBER 9xxxxx DESCRIPTION OF THE PROBLEM No signal.No band. This customer also uses ab51366 (Anti-PRPF8 antibody [2834C1a], mouse monoclonal antibody) to conduct with the same sample and got the target bands. SAMPLE • Species: Human • What’s cell line or tissue: PRPF8 –transfected A549 • Cell extract or Nuclear extract: cell extract PRIMARY ANTIBODY • Species:Rabbit • Reacts against: Human PRPF8 • At what dilution(s) have you tested this antibody: 1/1000 • What dilution buffer was used: 5％ milk in PBST • Incubation time: 3 hours • Incubation temperature: 25℃ • What washing steps were done: PBST wash 3 times,10 mins each time DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Yes , positive controls : transfect PRPF8 plasmid to cell. ANTIBODY STORAGE CONDITIONS -20℃ SAMPLE PREPARATION • What lysis buffer was used: RIPA buffer • What protease inhibitors were used: No • What loading buffer was used: 5X sample buffer (2-mercaptoethanol and glycerol with 10％ SDS in Tris HCl) • Phosphatase inhibitors : No • Did you heat the samples: temperature and time: 100℃ , 10 mins AMOUNT OF PROTEIN LOADED 250 ug ELECTROPHORESIS/GEL CONDITIONS • Reducing or non reducing gel: reducing gel • Reducing agent: SDS • Gel percentage : 6％ TRANSFER AND BLOCKING CONDITIONS • Transfer conditions: (Type of membrane, Protein transfer verified): Blocking conditions • Buffer:PBST • Blocking agent: milk what percentage: 5％ • Incubation time:1 hour • Incubation temperature:25 ℃ SECONDARY ANTIBODY • Species:Goat • Reacts against: rabbit IgG • At what dilution(s) have you tested this antibody: 1/3000 • Incubation time: 1 hours • Wash steps: PBST wash 3 times,10 mins each time • Fluorochrome or enzyme conjugate: enzyme conjugate • Do you know whether the problems you are experiencing come from the secondary? No. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES No signal is observed with ab79237, but ab51366 (ab against the same target) identifies a band of the correct size.

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab79237. I would also like to clarify a couple of details.

1. has the secondary antibody been shown to function appropriately?

2. Are you applying protease inhibitors in the lysis buffer?

3. Are the samples tested with the ab51366 exactly the same? What are the differences between the A, P, NS1, 1-93, 94-239 samples loaded? 

I would suggest increasing the concentration of the antibody, we have used 1 µg/ml with this antibody previously (1/500) but I would also try 1/250. I would also suggest incubating the antibody with the membrane overnight at 4°C and reduce the blocking content of the diluent (to 0.1%). I would also suggest using BSA as a blocking agent as this has been used in house to characterise this antibody.

Should the suggestions not improve the results, please do let me know, the applications for which you are using it for are using it are covered by the Abpromise and should the suggestions not improve the results. you would be entitled to a free of charge replacement or credit note.

I hope this information is helpful, and I thank you for your cooperation.  

Thanks for the quick reply. Yes, the WB conditions were identical to the conditions you listed. ATE1 is expressed in 293T cells at least based on microarrays (for example dataset GSE21092).

Let me know if we can try out the other antibodies.

ANSWER:

I suggest trying anti-ATE1 ab90561, which is a mouse monoclonal, tested for reactivity with human Jurkat but untested on mouse samples. One issue might be reactivity of an anit-mouse IgG secondary with endogenous IgG in the mosue lysate, but this is unlikely to be a problem with 3T3 cells. We do not have another anti-ATE1 rabbit polyclonal, or another lot of ab84636.

For PRPF8, I suggest a rabbit polyclonal, ab87433. This is tested against the same cell lines as ab79237 but differs in that it is raised against a peptide from the other end of PRPF8, the C terminus.

Please note that PRPF8 is a very large protein, and that efficacy of transfer may be an issue. Were you able to confirm that transfer was successful, for instance with a Ponceau Red stain? If it is not, then another antibody will not help. For large proteins, we recommend PVDF membranes instead of nitrocellulose, removing methanol from the transfer buffer, and including SDS to a concentration of 0.1%.

Please let me know what you would like. A credit or refund is also an option.

Hi, We recently purchased two antibodies (anti-ATE1 ab79237 and anti-PRPF8 ab84636) from you and they don't work at all Western Blots, despite the data sheets provided with them. We tested both antibodies with 293T and NIH3T3 lysates, and at least ab79237 is supposed to work with NIH3T3 cell lysates. Is there a way to try another antibody or are we forever doomed? Generally Abcam antibodies have worked really well for us, and these were the first antibodies that didn't work at all.

ANSWER:

We have two other antibodies against each of these targets which may give you a better result.

However, before sending replacements, we typically collect details of the protocols from our customers before issuing a replacement or credit/refund, in the hope that there may be a simple modification that fix the problem. The western blot protocol for each of these antibodies is standard, and I expect they are similar to what you tried.

In brief, 10 ug of lysate was loaded per lane of the gels. After transfer, the blots were blocked with 5% milk, and then incubated with 1 ug/ml antibody, then visualized with ECL. Can you please confirm that this is what you did? If not, please send the details of your protocol.

I do not know if expression of ATE1 in these cell lines is supressed; a quick search online showed some mentions of transfected 293T and 3T3, but none of endogenous expression. The antibody ab84636 was tested on HepG2, and another antibody, ab90561, was tested on Jurkat. Do you have evidence that 293T and 3T3 express ATE1?