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We have a customer trying to perform western blotting with the XIAP polyclonal antibody (Catalog Number ab2541). They could not obtain a specific band on the blot. They tried few protocol with room temperature incubation as well as overnight incubation with similar results. Blotting with other anitbody suggested the lysates are fine. Initially, what is your recommendation for primary antibody incubation condition? |
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ANSWER: |
Thank you for your enquiry. I am sorry to hear that your customer has been having difficulties with this antibody. Initially I would like to recommend that they apply the antibody at a dilution of 1:100 at 4oC overnight using 3% BSA/TBST as a blocking agent to determine whether the antibody is to blame. I hope this information helps, please do not hesitate to contact us if you need any more advice or information. |
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BATCH NUMBER 95586 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No staining (once) High background (twice) Weak signal even in negative control (twice) SAMPLE paraffin embedded human tissues PRIMARY ANTIBODY XIAP (AbCam); overnight incubation at 4?C; dilution 1:100, 1:200, longer washings with TBS-BSA 0.1% SECONDARY ANTIBODY Goat anti-rabbit (KPL); 45 minutes incubation, three times wash with TBS-BSA 0.1% DETECTION METHOD Streptavidine peroxidase followed by TBS-BSA 0.1% washing 3 times and then DAB-chromogen method for detection POSITIVE AND NEGATIVE CONTROLS USED Rabbit IgG used as negative control To the best of my knowledge there is no positive control tissue for IHC. ANTIBODY STORAGE CONDITIONS -20?C, aliquoted FIXATION OF SAMPLE Paraformaldehyde ANTIGEN RETRIEVAL Microwave boiling for 10 minutes in citrate buffer PERMEABILIZATION STEP Not used BLOCKING CONDITIONS Normal Goat serum (45 minutes) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? 1. Dakocytomation Antibody diluent with background reducing components 2. Washing with Tween to reduce background ADDITIONAL NOTES Can you supply a specific negative control for this XIAP antibody which I could try in IHC on paraffin embedded tissues?
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ANSWER: |
We have been informed by the source of ab2541 that western blots showed mouse intestine was positive for XIAP. We do not have a negative tissue sample available unfortunately. I hope this information will be useful, please do not hesitate to contact us if we can be of further assistance, |
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BATCH NUMBER 95586 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No staining (once) High background (twice) Weak signal even in negative control (twice) SAMPLE paraffin embedded human tissues PRIMARY ANTIBODY XIAP (AbCam); overnight incubation at 4?C; dilution 1:100, 1:200, longer washings with TBS-BSA 0.1% SECONDARY ANTIBODY Goat anti-rabbit (KPL); 45 minutes incubation, three times wash with TBS-BSA 0.1% DETECTION METHOD Streptavidine peroxidase followed by TBS-BSA 0.1% washing 3 times and then DAB-chromogen method for detection POSITIVE AND NEGATIVE CONTROLS USED Rabbit IgG used as negative control To the best of my knowledge there is no positive control tissue for IHC. ANTIBODY STORAGE CONDITIONS -20?C, aliquoted FIXATION OF SAMPLE Paraformaldehyde ANTIGEN RETRIEVAL Microwave boiling for 10 minutes in citrate buffer PERMEABILIZATION STEP Not used BLOCKING CONDITIONS Normal Goat serum (45 minutes) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? 1. Dakocytomation Antibody diluent with background reducing components 2. Washing with Tween to reduce background ADDITIONAL NOTES Can you supply a specific negative control for this XIAP antibody which I could try in IHC on paraffin embedded tissues?
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ANSWER: |
I'm sorry to hear you are having problems with ab2541 in IHC. As we have not tested the antibody in this application it is very difficult to find out what the problem might be and I am waiting to hear back from the source of the antibody regarding positive and negative controls to help you. I would like to suggest to consider different antigen retrieval methods (e.g. enzymatic) and adding triton x100 (0.3%v/v) in the buffer used to dilute both primary and secondary antibodies to maximise permeabilization and penetration of the antibodies. I will forward to you the information from the source of the antibody as soon as I receive it, my apologies for the delay, Thank you for your patience,
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can you tell me if this antibody cross reacts with rat and will it IP? |
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ANSWER: |
This antibody has not been tested on rat or IP so we have no further information on this. |
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please can you send me the antigen peptide sequence that has been use to raise this antibody thank you |
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ANSWER: |
The immunogen is a 12 a.a. peptide around residue 240 of human XIAP. However, we are unable to disclose the detail sequence, as it is proprietary. |
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