Protein precipitates - (Aug/07/2006 )

I try to purify my protein throuh affinity column but i have some non specific binding which is higher molecuar wheight then my protien wheh i elued through elution buffer.
Theni tried to increase the sodium chloride concentraion NaCl up to 2M i have now clear sediments at the bottom of the tube. I think its a sodium chloride preciptates or some thing else.
But i donot think so that is NaCl becasue in my buffer sample NaCl is not precipating while in my wash and elution buffer with protein sample i have some transparent sediments. What it can be.
suggest me.
regards
samita

this is a puzzlement...

OK. increased NaCl should increase solubility. why did you add it in order to increase binding specificity? I'm not clear on that? and if NaCl increases solubility, you should not be getting precipitation of your protein...what are the other buffer components? what is the pH of your buffer, and have you checked it recently? and, is this a his-tag purification? that is the method that I know the best, I may not be able to help you with specifics if it's another method

there are other tricks to increasing binding specificity.

Yes its His tag affinity through cobalt column. My proteini size is almost equal to bacterial house keeping protein and i have lot of other bands in my elution fraction and my elution bans is also not appreciable. I have NP-10 buffer of Ph 8.0 and its recepie is 50mM NaH2PO4, 300mM Nacl, 10 mM Imidazole, its my lysis and caliberation buffer,
for washing i am using 20 mM imidazole and for elution 250 mM imidazole.
But i weak binding and other bands in elution fraction.
so i increase NaCl concentation to see what happen this time i have some sediments at the bottm of fraction in wash and elution sample.
regards

I think it would be a good thing for you to follow this link
and read the sections about troubleshooting and about binding specificity. even if you are using a different system, I am guessing most of the chemistry is the same and their advice will still be useful to you.

I GET DIFFERNECE OF BINDING WHEN I DID MY STEP AT LOWER TEMPERATURE. LOWER TEMPERTURE SLIGHTLY INCREASE BINDING. TRY TO ENJOY SUMMER IN COLD ROOM.


quote name='samita' date='Aug 8 2006, 04:01 AM' post='62956']
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[quote name='spanishflower' post='62931' date='Aug 7 2006, 11:35 PM']
[quote name='aimikins' post='62855' date='Aug 8 2006, 02:46 AM']
this is a puzzlement...

OK. increased NaCl should increase solubility. why did you add it in order to increase binding specificity?

i am confused now as my professor asked me to increase the NaCl concentration in my buffer to help in increasing binding capacity? i am doing GST tag purification????

there are other tricks to increasing binding specificity.

can u share it with us?
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spanishflower, follow the link I posted, read through the troubleshooting, and you will get tips to increase binding specificity (or at least things to try)

well, as to whether it will help more protein bind to your column, I suppose if more protein were in soluble form you'd get better binding? I'm really not sure why he told you that? I know that when I was purifying a handful of proteins that were very prone to precipitation, but I needed native protein if it were possible, one of the recommendations (also in that link) is to increase the NaCl up to 2M in all your buffers. Which I did, and got much better yield and no precipitation. also had to add a little glycerol, but the point is that increasing your NaCl increases protein solubility, so I don't know why he told you to add it? I'm not saying he's wrong, I just don't know why it would help? unless insoluble protein just doesn't bind well and so allows for more background binding?

thankx amikin
i asked him to be more specfic about this and he said that increasing concentration of NaCl will decrease the non specfic binding of my ( protein 2) to the sepharose beads
i am doing pulldown assay using GST taged protein 1 and testing its interaction with protein 2