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Bradford assay and erythrocytes

Bradford assay and erythrocytes - Protein Science

Bradford assay and erythrocytes - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 04-07-2010, 12:17 PM
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Default Bradford assay and erythrocytes



The situation is as follows. I have lysates of several cell types (TBS, freeze-thaw cycles, nothing special in buffer), determine the total protein content by Bradford, take 0.5 mg/ml of total protein for cleavage analysis of another purified protein, put it all on gel and see with Western if any cleavage occured.
So far so good, platelet and megakaryocyte lysates can be determined nicely, also on gel by CBB staining we can see that the matching of protein content worked.
But not erythrocytes. With them I get a huge overestimation of protein content, had to dilute the lysate 4000 times to get the readout within the standard curve. And that results in ~100-150x higher estimated protein content than other samples. And when I put the samples of gel, the lane of erythrocytes is empty. Tried to put 100x more concentrated erythrocyte sample on gel, got a big blue lane (i.e., too much protein). Of course, I can go on running gels with various dilutions of the erythrocyte sample till I have a lane that is comparable with other cell lysates but I'd like to be able to quantify it properly.
So the question is - does anybody know what could interfere with Bradford analysis in my erythrocyte sample. Anbody heard of haeme or haemoglobin giving problems in Bradford analysis? Any other suggestions how to quantify the total protein content in erythrocytes also would be welcome.
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  #2  
Old 02-26-2013, 05:23 PM
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Default Re: Bradford assay and erythrocytes

Quote:
Originally Posted by bloo_1 View Post
The situation is as follows. I have lysates of several cell types (TBS, freeze-thaw cycles, nothing special in buffer), determine the total protein content by Bradford, take 0.5 mg/ml of total protein for cleavage analysis of another purified protein, put it all on gel and see with Western if any cleavage occured.
So far so good, platelet and megakaryocyte lysates can be determined nicely, also on gel by CBB staining we can see that the matching of protein content worked.
But not erythrocytes. With them I get a huge overestimation of protein content, had to dilute the lysate 4000 times to get the readout within the standard curve. And that results in ~100-150x higher estimated protein content than other samples. And when I put the samples of gel, the lane of erythrocytes is empty. Tried to put 100x more concentrated erythrocyte sample on gel, got a big blue lane (i.e., too much protein). Of course, I can go on running gels with various dilutions of the erythrocyte sample till I have a lane that is comparable with other cell lysates but I'd like to be able to quantify it properly.
So the question is - does anybody know what could interfere with Bradford analysis in my erythrocyte sample. Anbody heard of haeme or haemoglobin giving problems in Bradford analysis? Any other suggestions how to quantify the total protein content in erythrocytes also would be welcome.
I have the same problem.
I tried to quantify the proteins extracted from RBCs, but I could not get neither the curve. Bradford needs the extraction buffer to make standards dilutions, and since I used cold deionized water, the curve was terrible.
Any suggestion?

Thanks in advance
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Old 03-06-2014, 07:18 AM
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Default Re: Bradford assay and erythrocytes

Try to eliminate haemoglobin interference
Previous experience employing organic solvent mixture (ethanol:1-butanol - 60:40 -v/v) starting by dilution lysates 1:1.087, or using cationic metal such as zinc chloride (5 mM) or zinc acetate (4 M) is obtained a precipitate of all haemoglobin molecules and clear supernatante containing remaining proteins of the lysates
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