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Product code: 71333
Lot number: 931321
Inquiry: Hello, I have had troubles with the specificity of the Talin-1 Antibody (ab71333, purchased ). Conditions and protocol as follows: - Loaded 20ug protein - SDS-PAGE was used, a standard running buffer was used, run at 120V for ˜2 hours. - Transblot Turbo from Bio-Rad was used for transfer. This used standard semi-dry consumables following the protocol set out in the Transblot manual. The time of transfer was reduced to 20 min as we had previously found that 30 min lead to proteins being transfered to the filter paper. - Blocking buffer was 5% BSA in TBST for 1 hour at 4 degrees - Primary antibody was at 1ug/ml (recommended) in blocking buffer, incubated overnight (˜18hours) at 4 degrees - detected using fresh HRP-conjugated secondaries (known to be working) at a concentration of 1/3000 in blocking buffer for 1 hr at 4 degrees - 3x 5 min washes with TBST occurred between primary and secondary incubations as well as after secondary incubation. Membranes were kept in TBS after final wash. In the sample image uploaded the first lane (juxtaposed on the image) is a MW standards ladder, followed by 4 lanes of RWPE1 cell lysate, and 4 lanes of PC3 cell lysate. There is a very faint band at just above 250kDa, which is possibly Talin-1, however there are multiple bands further down the membrane which are much darker (for example: ˜60kDa, ˜40kDa and ˜35kDa). This appears to be a specificity issue as the 270kDa band should be the most defined band. Other factors (eg concentration of antibodies) appear to be fine as other bands are showing up. Any suggestions would be welcome.
Asked on Aug 13 2012
Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.
I have read the details you have kindly provided and have following further questions for better understanding of the problem;
- Which lysis buffer was used? Was it ice cold whencells lysis was done?
-Did you try any sort of protocol troubleshooting?
- Was the lysate heated at 100C for 5 minutes?
- Could you please re-attach the image?
- Have you added any protease inhibitor in lysis buffer?
Thank you very much for your cooperation. I will look forward to hearing from you soon.
Answered on Aug 13 2012