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Thank you for getting back to me. Sorry for the delay in getting this information to you. We have actually been testing a third antibody from you that is also giving us the same problem - anti–GPSM2 antibody (a b84571). Hopefully,
you will be able to assist us.
Below is the information you requested. I've included the information as well for the anti-GPSM2 antibody.
1) What was your original order or PO number?
Anti-spastin antibody (ab31850) - ***
Anti-FIGN antibody (ab122238) - ***
Anti-GPSM2 antibody (ab84571) - ***
2) Could you please provide a description of the problem (no signal, high background, incorrectstaining, etc) or an image of your results?
SPASTIN – many bands of incorrect size. None of the observed bands disappeared or decreased in intensity in cells transfected with siRNAs against Spastin.
FIGN – no signal
GPSM2 – no signal
The mRNAs for all these genes were detected by RT-PCR in the cells that were used for the WB.
3) What type of samples were you testing?
Which species were they from, what tissue type or cell line, and how were they prepared?
For example, what lysis buffer was used in WB, were the samples reduced and denatured, and how much protein was loaded?
For IF, how were the cells fixed and permeabilized?
Proteins extracts from RPE-1 (human), HeLa (human) and NIH3T3 (mouse) were used to test the FIGN and SPASTIN antibodies.
Proteins extracts from HeLa (human) were used to test the GPSM2 antibody.
The protein extracts were prepared with the following lysis buffer: 50mM HEPES, 100mM KCl, 0.3% NP-40, 10% glycerol, 1mM EGTA, 1mM MgCl2 + protease inhibitors and
The protein extracts were quantified by the Bradford assay and 25mg were loaded/lane in 10% SDS-PAGE after
being incubated at 95ºC for 5 min.
4) What were the dilutions and incubation times for the block, primary, and secondary antibodies?
What blocking agent and secondary antibody were used?
The proteins were transferred to PVDF membranes. The membranes were blocked for 1h in 5% Skimmilk in TBS-Tween. The membranes were incubated with the primary antibodies in
blocking solution over night at 4ºC with constant agitation. For each antibody the following dilutions were tested: 1:100; 1:250; 1:500; 1.1000.
After the incubation with the primary antibodies the membranes were washed 3 times for 10 minutes with TBS-Tween. The membranes were then incubated with HRP- conjugated secondary
antibodies for 1h at room temperature. After this incubation the membranes were washed 3 times with TBS-Tween. Finally the membranes were incubated with ECL and exposed.
If you require any additional information please let me know. Any help you can provide would be great.
Asked on Jul 17 2012
Thank you for your reply.
For the Spastin antibody ab31850, could you please provide an image of your blot or let me know what size bands you are observing? Spastin has several known isoforms that could account for multiple bands between 54-67 kDa. Regarding the lack of change with transfected cells and siRNA, can you tell me if the transfected Spastin was full length and from human? What siRNA were you using, and was the knock-down validated by RT-PCR?
For the GPSM2 antibody, the protocol you provided seems fine and we would definitely expect you to get good signal using this method in HeLa cells by WB and ICC/IF. I would be happy to offer you a free of charge replacement, credit, or refund for this antibody.
The FIGN antibody ab122238 has not been validated for use in WB or ICC/IF on cultured cells. Have you tried this antibody in IHC-P? It is possible that it may work in WB or ICC/IF, but we do not have any information about how the antibody would work for those applications. To increase your signal, you could try preparing your samples with RIPA buffer, which is more effective at releasing nuclear proteins. You could also try increasing your ECL exposure time or your secondary antibody concentration.
I hope this helps, if not, please let me know and I will be happy to help you further.
Answered on Jul 17 2012