Anti-active Caspase 3 antibody (ab13847)

Enclosed you may find pictures after testing according your suggestion.
Still antibody works wrong.

ANSWER:

Thank you for your reply.

I am sorry that my suggestions did not improve your results with this antibody. I would be happy to offer a replacement or a refund. Please let me know which you would prefer.

I apologize for this inconvenience. I look forward to your reply so that I may assist you further.

I got very nice bands in the expected size with Iba-1 Western blot from the cell culture samples which were treated with dithranol. It is true that the tendency what we wait is not correct, ( so not the wait differences in the amount of the protein) , but we got the band at a correct place.
The antibody was ordered at 2010, it was aliqouted at -20 C.

ANSWER:

The only explanation I have, assuming the samples are intact and effectively reduced, is that the antibody is failing. It should be detecting a fragment at 17 kDa (not 12 kDa; that was a mistake on the datasheet).

However, one of your earlier blots did show a band at 17 kDa, and it appeared only after treatment, if I understood you correctly.

I think the band at 75 kDa is irrelevant, but I do not know why there is so much of this protein, as revealed in the Ponceau stain.

Have you considered looking at any other markers of apoptosis, for instance PARP, or are you only interested in caspase 3?

Unfortunately, your order is past the limit of our guarantee, which is in effect for 6 months following purchase. If you are interested in buying a different antibody, I will be happy to help.

Thanks your suggestions, we will try the sample buffer as you suggested. We treated the cells with dithanol, which cause very hard oxidative stress. In fig 2 the samples were we got bands were treaed with dithranol the other samples were not. I know that staurosporine is the main apoptosis inducing reagent, but unfortonetly we have not got any in our lab.

ANSWER:

I am glad to help. I think figure two suggests that the treatment and antibody are effective. Hopefully your blots with new samples will not have the high molecular weight signal. Please let me know how it goes.

Want to use antibody in IHC-P on Pig tissues.

ANSWER:

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Expiration date: 18-05-2012

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The samples were primer neuronal culture isolated from 18 days old rat embryo, cultured for 7 day long, than the cells were collected, precipitated in a lysis buffer (50 mM Tris pH 7,5, 150 mM NaCl, , 0,1 % Nonidet -P-40, 0,1 % colic acid, 2 ug/ml leupeptin, 2 mM PMSF, 1 ug/ml pepstatin, 2 mM EDTA) ,before loading the gel the samples were diluted with sample buffer ( 0,05 M Tris pH 6,8, 10 % glycerol, 2% SDS, 5% 2-mercapthoethanol, bromophenolblue) in 2:1 ratio, the samples were boiled for 5 minutes, then loaded to the gel, which contains 10% acrylamide, 1,5 M Tris pH 8,8, 10 % SDS, 10% APS, and 10% TEMED, after running and transfer the nitrocellulose membrane were blocked overnight with 5 % bovine serum albumine containing TBS -Tween, and then your antibodies were loaded for 2 hour at 1/1000 dilution in 1% BSA containing TBS -Tween, after washing, the secondary antibody were added to the membrane for 1 hour, then the signal were developed with ECL system. I attached the film of the experiment (Film 1).

Before this experiment I tried to use your antibody in samples from rat trigeminal ganglia several times and several protocols, I got bands at 17 kDA if I used the same protocols as I mentioned (Film 2), but it is true there are higher bands also but no 12 kDA bands.

If I used another protocol with ganglional samplesI got correct bands (17 and 12 kDA) but I got these bands even in the non treated samples and no band in 30 kda region were the non cleaved caspase should be visible..... In this experimentI used 16% gel which contains 10% glycerol and I used also different blocking and fixation steps, In this case after transfer I fixed the membrane with 0,2 % glutaraldehyde then blocked with 1M lyzine for 1 hour, and 5 % containing BSA also... (film 3) Do you have any other experience with this antibody?

Both samples (the cell culture and the ganglional also) add very nice valuable band with Iba-1 ( ionized calcium binding antibody which is specific for monocyte origin cells) polyclonal antibody with the first mentioned protocol.

I treated the samples with a solution which cause very strong oxidative stress.

The lot number of the antibody is : 736451

ANSWER:

Thank you for sending the details of your samples and protocol.

The band you see in film #2 at 17 kDa is expected. I have contacted the laboratory to explain the note stating that the 12 kDa band has been detected too. Based on the location of the immunogen, this note appears to be a msitake. The pro-form at 30 kDa should also not be detected. The antibody was developed to be specific for the 17 kDa fragment.

Film #2 (trigeminal ganglia) seems to conform most closely to what we predict for this antibody, except for the higher molecular weight smear above the 17 kDa band.

Regarding the lack of differences in band intensity between your treated samples and the untreated samples, do you observe any evidence of apoptosis that demonstrates the efficacy of the treatment?

Have you stained blots of these samples with antibodies for any other proteins aside from this antibody and Iba1? If so, were the results what you expected? I am a little concerned about the composition of the sample buffer you specified, as the components appear to be half of what most recipes call for. If I understand your protocol correctly, the 2X sample buffer has 2% SDS and 5% beta-mercaptoehtanol. When you add the samples, these concentrations are 1% and 2.5% respectively. Is that correct?

I look forward to your reply. If I cannot offer a suggestion for a protocol modification, I will be happy to replace the antibody.