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I loaded 2.5mg/ml of total protein (the original concentration) I also tried lower concentrations (0.5 mg/mL and 0.25 mg/mL) and saw signal only in the PC12 nuclear lysate of corresponding concentrations. I assume that if the PC12 lysate showed a clean signal at 2.5mg/mL the HeLa lysate should also work - but it did not.
Asked on Jan 20 2012
Thank you for your reply.
I appreciate your patience in this matter, but when you say you loaded 2.5mg/ml of total protein, how many microlitres of lysate is that? I ask, as we send 100ug of total protein at at concentration of 2.5mg/ml (therefore we you should have received 40ul total volume). I just want to make sure that the problem is with the lysate and not that more needs to be used. Although you used both the HeLa and PC12 lysates at the same concentrations, it may be that you need to use more of the HeLa lysate, as you were seeing a signal for Tubulin (the signal was weak, but it was present).
Once again thank you for your patience in this matter, if it does turn out that there is a fault with the lysate, then I would be more than happy to send a new vial.
I look forward to your response.
Answered on Jan 20 2012