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I’ve attached the protocol I used for the Western blot. I’m trying to find a good antibody to HCV NS5A so that I can select stable clones overexpressing the protein. So far, I haven’t been able to pick up any NS5A using two proteins from Abcam, ab49033 and ab68096, and high concentrations (1:100, 1:250) of four different antibodies (ab20342, sc-57776, ab20773, ab2594).
Asked on Jun 20 2012
Thanks for sending along your protocol.
1. ab49033 is unlikely to be detected by these antibodies as it only contains about 100 aa of the total protein. As described on datasheet this product containsamino acids"corresponding to amino acids 2212-2313 of Hepatitis C Virus NS5" The epitopes recognized by these antibodies would have to recognize NS5A within this small region to work.
2. Usingponceau S stain post transfer, have you been able to determine protein is present on membrane after transfer?
3. Regarding your protocol, a) Boiling of samplesis not recommended; instead heat at 70 deg C for 10min prior to loading gel b) Try blocking with 5% BSA instead of milk c) Use 1:1000 for primary antibodies and 1:5000 for secondary antibodies.
I look forward to hearing if these suggestions prove useful.
Abcam Scientific Support
Answered on Jun 20 2012