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Please read customer reply:
1. I can suggest to try RIPA rather than Tris buffer to lyse the samples. This may provide a more suitable protein preparation. - we used this buffer for lysis in the past, it provided no great advantage for lysis. In any case, this should not change the results of the western analysis.
2. Try the antibody at a higher concentration to increase the signal, for example 1:1000 or even 1:500. Incubating overnight at4oC can often provide more specific and efficient staining. - the lowest dilution we tried was 1:2000, but we did extend the incubation time to 48 hours at 4degC. Our positive control should have yielded a band under these conditions.
3. Has the transfer to the membrane and quality of the sample been assessed using a loading control? yes.
4. Is the current vial of secondary antibody working well with other primary antibodies? yes
Asked on Aug 02 2012
Thank you for your message and for providing kindly this further information.
I appreciate the time you have spent on these experiments and it is regrettable the results have not been successful. I would be pleased to arrange a refund or credit note in compensation.
In addition to this, experiments can often require individual optimization, andI suggest it would be beneficial to consider trying RIPA lysis buffer and a higher concentration of antibody for future experiments. Optimization such as this can sometimes help to improve results.
I look forward to hearing from you with details of how you would like to proceed in this case.
Answered on Aug 02 2012