I transfected HEK293 cells with constructs containing an RFP reporter. The signal was so strong that the RFP was visible to the naked eye when I pelleted the cells. And the protein lysate even had a pink tint to it. I used Pierce BCA Assay kit to determine a protein concentration and ran my western. The Actin bands were way off! The condition (that happened to have a much stronger RFP signal) showed reduced actin compared to the other condition.
Should I try a different way to measure protein concentration? Or a different loading control?
Could the BCA be off due to the RFP in the lysate? Could it be giving me a higher concentration reading than what it actually is?
The BCA assay is measured at 562 nm and the signal of RFP comes at 588 nm, so if you can see the product in your lysate you will have a prominent overlap.
Bradford is no alternative because it´s analyzed at 595 nm. You can try Biuret with extinction at 546 or Lowry with 650 or 750 nm (according to Wikipedia).
You could also do a measurement of Abs at 280 nm. I like Mark's answer, but this would be a nice check: both folded and denatured protein should give the same A280.
Thanks for the feedback everyone! What I'm hearing from several sources suggests that the RFP is causing a problem. With your input plus that of tech support, I'm going to try Pierce 660nm protein assay.
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