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His-tag protein vs. BSA

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#1 Yasser Almeida Hernández

Yasser Almeida Hernández


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Posted 28 April 2011 - 10:30 AM

Hi all...
I'm purifying a 105 kDa glycoprotein from mammalian cell culture using Cu. As know, the mammalian cell culture often require a percent of fetal bovine serum wish contain huge amounts of bovine serum albumin (BSA). I'm using imidazole at 2, 10 and 100 mM in the elution buffer and in the all cases my protein and the albumin co-eluted with my protein as well as other minority contaminant proteins. I know that the Ni (Ni-NTA) in the most commonly used ion i this type of purification but I don't have the Ni-NTA column neither the salt itself right now.

The BSA have 17 histidine along all the sequence, but, as the quantity is so high, the protein stick to the column, i think...

So, do you have any suggestion to eliminate the albumin in the purification? That is normal?

Thanks all in advance...

#2 proteaMatt



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Posted 28 April 2011 - 01:58 PM

What is the volume of your sample that you want to purify? Do you know the approximate mass of the BSA in your sample?

An albumin depletion kit can deplete 90% or more of the albumin from your sample so long as it falls within the parameters of usage. Usually, I recommend samples of <100 µL that contain up to 500 µg of albumin. I have found it to work really well on a 10% fetal bovine serum growth media, so my recommendation would be to harvest your cells as normal and then deplete the albumin from your sample before processing it with your Cu. The other thing to keep in mind is that following albumin depletion the sample will be buffered at a pH of around 7 with a phosphate buffer. So make sure that wouldn't interfere with your following steps.

Edited by proteaMatt, 28 April 2011 - 02:01 PM.

Lab Technician at Protea Biosciences

#3 protolder



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Posted 28 April 2011 - 10:13 PM

Hola, in all the methods low concentration of imidazole about 10-20 mM are added to bound buffer and wash buffer in order to eliminate the inespecific interactions, washing this inespecific bounded protein and remaining bound 6His tagged proteins wich eluted with high imidazole concentrations. Your case is extrange could be a specific interaction of your protein with BSA? because the affinity of BSA and the 6HIS tagged protein have to be different, or the pH of your buffer is too low? avoiding the bound . Buena suerte

#4 Yasser Almeida Hernández

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Posted 29 April 2011 - 07:49 AM


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