Hello,
I have been performing Western Blots to study the protein levels of a rodent skin and muscle sample. However, I have not been able to produce homogenous reference bands.
I suspect that this is due to the presence of blood in the samples interfering with absorbance measurements. To provide more context, my sample is a surgical site harvested not long after surgery, and I have noticed varying red colourations in protein lysates between samples. My reference gene bands for another sample type (not skin and muscle) with a pale protein lysate are fine.
I have repeatedly performed the Bradford assay, every time before i run a new gel; I have also attempted saline perfusion after euthanising the animal, but even when the liver turns pale the surgical site still appears bruised and patchy with blood. In addition, I have tried washing my sample with saline; and switched between difference reference bands (B actin and GAPDH). I have tried everything I can think of but nothing seems to work for me.
Is there a way to remove haemoglobin from protein lysates? Or is there a more reliable way to test for protein concentrations?
This has been such a head-scratcher... I would be very grateful if anyone could give me some advice. Thank you very much in advance!
Jen C. I assume you are using a protease and phosphatase inhibitor cocktail for the preparation of tissue homogenate. Include 1% of the inhibitor cocktail in the tissue buffer, homogenize or sonicate as per requirement, and centrifuge at 12000 rcf for 15 minutes. Following that collect the supernatant using a syringe. Also for protein estimation, you can opt for the BCA reagent, as it gives more consistent results than the Bradford reagent.
Hi, Kindly check this - https://www.researchgate.net/post/How-can-I-remove-hemoglobin-from-protein-lysates. And for protein concentration BCA Assay: The Bicinchoninic Acid (BCA) assay is less susceptible to interference from hemoglobin and can provide more accurate protein quantification. Good Luck.
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