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Thanks for your mail. I used two anti-WT-1 antibodies (ab15249 and ab64681) and both are giving multiple bands with my experiments. The ab64681 is giving one huge band at 100 KDa along with other additional bands that are alsotec observed with ab15249.I wrote my last mail as you asked forfeedback onyour antibodies.When I compared I find ab15249better than ab64681 with my experimental system, although it also produced multiple bands.I am still confused with the occurrence of these bands.
Asked on Feb 06 2012
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After reviewing the details of your contacts with my colleagues, I have only located the image of ab73466 with ab64681. It appears that when running the peptide ab73466 in SDS-PAGE, you detected a band around 75 kDa. While ab73466 is suitable for blocking the specificity of ab64681 after preincubation, it is only 18 amino acids in length and is therefore too small to be detected in SDS-PAGE.
This peptide is 70-90% pure and is supplied in a buffer that does not contain any additional proteins. Because this protein is synthetic, the 10-30% contamination will be amino acids, chemicals, and partial peptides, none of which would be expected to run at 75 kDa. The The presence of the 75 kDa band indicates that there may be some protein contamination in the loading or running buffer. This may also help to explain some of the additional bands in your western.
According to the UniProt database, there are 8 isoforms of Wilms Tumor Protein with molecular weights ranging from 34 - 57 kDa. This protein is also known to interact with other proteins and to form homodimers (which may be the band observed at 100 kDa). Using a buffer with ionic and non-ionic detergents (such as RIPA buffer) and boiling the protein in the presence of BME or DTT will help to ensure that all multimers are broken down.
I hope this helps, please let me know if you need any additional information or assistance.
Abcam Scientific Support
Answered on Feb 06 2012