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Friendly antibody buffer to prevent precipitation?

antibodies buffers precipitation organic chemistry

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3 replies to this topic

#1 Michael Starr

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Posted 24 March 2016 - 09:18 AM

We did an affinity purification for antibodies against a drug in our company. The column was very efficient, so we got a lot of antibody off of it. However, the other side is that the antibody (though not that concentrated, maybe 500 ug/mL) partially precipitated out of solution. The buffer we use is DPBS. Are there tweaks that can be made to the buffer that would prevent this crashing out in the future? What contributes to this phenomenon? My chemistry background isn't the strongest. I know you can change the salt concentration, maybe use a different primary buffer constituent (sodium bicarb, tris, etc. instead of phosphate). Different pH. Carrier protein (though that would mess with the sample purity). Anything else?



#2 mdfenko

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Posted 25 March 2016 - 03:26 AM

have you tried to resolubilize the precipitated protein?

 

if so, were you successful?

 

precipitation may be caused by many factors including: pH, salt concentration, presence or absence of detergents, presence of organics, protein concentration, denaturation, or a combination of any or all.


Edited by mdfenko, 25 March 2016 - 03:27 AM.

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#3 Michael Starr

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Posted 25 March 2016 - 09:20 AM

It was actually my coworker who was in charge of this. I think he tried to resolubilize and it didn't work. But the loss, according to a total protein BCA before and after precipitation, was not too bad. We are thinking of using a different buffer--bicarb instead of PBS e.g.--or maybe add a little Tween 20. I did some research and it looks like there are tons of things to try, so I'm sure we'll figure something out. Urea, kosmotropes (sulfates), other stuff.



#4 mdfenko

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Posted 28 March 2016 - 03:22 AM

urea will denature protein, as will some other additives. you have to be careful with what you use to ensure solubility.

 

it's probable that the precipitated protein is denatured and aggregated. this is not uncommon when purifying proteins. if you have enough viable antibody then i wouldn't worry about minor losses at this stage.


talent does what it can
genius does what it must
i do what i get paid to do





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