Protein G problem - Antibodies absent from both flow through and fractions (Feb/22/2007 )
Hello,
I seem to have run across a problem in my Protein G purifications. Recently, I was purifying a rather concentrated sample of PSA antibodies (10mls, 6.09mg) and thus I was instructed to use a 1ml Protein G column and purify this 10 ml sample in volumes of 2ml (thus over the course of 5 days). On day 1, i received a rather small amount of protein, then on days 2 and 3, I achieved quite high amounts. Then on day 4, I received nothing. I kept the flow through, and analysed it for IgG content, and again, nothing was found. So essentially, I received no antibodies in my fractions, nor in the flow through. Could it be possible that my use of Glycine-HCl to elute the antibodies is the problem? I have used Glycine-HCl solutions before with no problem, but I cannot as yet understand why this would happen so suddenly, especially since Protein G columns are to be used concurrently, and any decline should be gradual.
Many thanks.
Superfoot.
On day 1, i received a rather small amount of protein, then on days 2 and 3, I achieved quite high amounts. Then on day 4, I received nothing. I kept the flow through, and analysed it for IgG content, and again, nothing was found. So essentially, I received no antibodies in my fractions, nor in the flow through.
Superfoot.
What about your elution conditions? 0.1 Glycine pH 3.0 or another? Could you explain one more what does it mean your day 1, 2,3,4 chain isolation? Is sample were the same or different in Ab content? Did your analyse sample before purification on SDS PAGE due to estimate Ab content?
On day 1, i received a rather small amount of protein, then on days 2 and 3, I achieved quite high amounts. Then on day 4, I received nothing. I kept the flow through, and analysed it for IgG content, and again, nothing was found. So essentially, I received no antibodies in my fractions, nor in the flow through.
Superfoot.
What about your elution conditions? 0.1 Glycine pH 3.0 or another? Could you explain one more what does it mean your day 1, 2,3,4 chain isolation? Is sample were the same or different in Ab content? Did your analyse sample before purification on SDS PAGE due to estimate Ab content?
Elution conditions: 0.1 Glycine-HCl pH 2.7
As the sample itself is 10mls, I was instructed to perform the purification of the sample in stages, taking 2mls of the sample to purify, hence on separate days, I would be purifying a 10ml sample, taking out 2mls at a time. The sample is the same Ab content throughout all purifications, and no, I did not personally analyse the samples on SDS PAGE, as there is another lab responsible for that, who package their samples, and send them to my lab, from where I do the purifications.
You received no antibody from the total of 4 days of purifications or just on the fourth day?
How do you store your 2 ml samples ( I mean aliqote and keep frozen) or add protease inhibitors and store +4? Do you use protease inhibitors during chromatography process ( PMSF or another?) It is important because your protein G susceptible for protease attack. What is your binding conditions ( I mean binding buffer?) How old are your Protein G column and at what storage conditions do you use for your column?