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Please find attached one of the blots probed with HCP-1 Gt.
these are hela cell lysates of 30ug per well.
The same blot was probed for HRG-1 by protein of interest and also beta actin both of which were fine.
The Goat secondary is routinely used in the lab by other members.
The washing conditions are 2X TBST for 5 min, 1X TBS at RT after primary and after secondary.
Samples are washed in cold pbs, lysed in RIPA buffer with Protein inhibitor cocktail for 30 mins before being centrifuged @ 14,000rpm for 15 min, protein levels quantified using bradford reagent. Samples are ran on a 4-20% gel at constant amps. Transferred for 1 hour at constant amp also before blocking at RT in 5% milk TBST.
Asked on Oct 08 2012
Thank you for your reply with further information.
Reviewing the protocol and testing conditions, I do not think that further optimization will improve the results signficantly. You may of received a faulty vial or batch, and I must apologize for any inconvenience this has cuased. We have another HCP1 antibody that is suitable for human samples in WB:
ab98043 Anti-HCP1 antibody
Would you like to test this product or would you prefer a full refund? Please let me know how you would like to proceed.
Answered on Oct 08 2012