which kind of cell lysis buffer should I use?
Wenbo Wang
Saturday, 14 March 2009 03:02 UTC
recently,I’d like to isolate one subunit of a big protein complex-26 S proteasome.I don not know which kind of lysis buffer should be employed here as the complex should be disrupted to facilitate the isolation procedure in this assay whereas the denaturation of the subunit are not expected.Is there anyone could be kindly enough to offer me a protocol or something like that?
thank you in advance.
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Replies
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Hi,
Your choice of lysis buffer will be affected by many factors. Although I have never worked with this complex, there is a wealth of published information on this topic that should guide you well.
You will want to keep the following in mind:
1) Since you want a single protein you are not worried about denaturing the (proteasome) complex… in fact, you hope to release your component of interest from the complex.
2) However, you must choose conditions that allow you to capture the component you are interested in… so, stringency (or at least buffer composition) is an important factor.
How do you intend to capture the protein of interest? Affinity capture? Which tag will you use?
When you have answers to these questions, then it will be easier to give more advice.
Cheers,
John -
Hi,
Thank you for your reply.
I want the subunit I’m interested in to be purified by affinity purification. And the tag I am willing to use is biotin. I find lots kinds of lysis buffer are nondenaturing, which is proper and applicable to CO-IP. Unfortunately, they are out of my choice. The lysis buffer should be destructive to quaternary bonds between subunits while gentle to tertary and secondary bonds. The amount of the denaturing components,SDS or other kinds of denaturing agent,in the buffer may be a key question to be considered. -
Hi there…
I guess you are using some in vivo biotinylation method?Indeed you hit the nail on the head… you essentially want to know how much denaturing agent can the biotin/avidin can resist.
I dont have the straight answer for you, you may have to do some initial testing… In my experience lesser (but strong) tags such as the Protein A / IgG interaction can take 1M NaCl or more… even up to 2M urea.
But you will have to determine this for your tag, on your resin type and pH…
I have not had success using SDS as a component of a buffer… at most concentrations (even very low) I always see losses from the column. On the other hand, the detergent Sarkosyl is a good option… it is not as strong as SDS, but has similar properties (it is a medium strength ionic detergent).
Basically, you want to use chaotropic salts or ionic detergents… you just need to find the strongest condition that still works..
I have an avidin / biotin handbook (in pdf form, ~360 pages, I think it was compiled by Pierce) I can send to you in case you might find it useful… however, I cant be much more help on this subject since I dont have much experience working with these particular as affinity reagents.
Send me your direct email through this site and I will reply with the document attached.
Cheers,
John -
Hi Jone,
Thank you for your reply again.
You have figured out what I want to do in my assay.However, it is somewhat more complicated.
I have constructed a biotinated compound which is specific for the subunit in the protein complex.Firstly,I would like to saturate the beads with this biotinated compound.Then, the whole cell lysis will be applied onto the beads.Ideally,the subunit will be fished out by the biotin tag.So,the key question is that the subunit should better maitain its higher structure especially at its active site which the compound can recognize.
Fortunately, I have found some denaturing lysis buffers, such as RIPA,can be employed to disrupt the protein protein interaction,whereas the higher structure of the subunit protein may be also influenced.
And finally I make the conclusion that there is an overlap between the quaternary bonds disruption and the tertary and secondary bonds disruption,because they are comprised of hydrogen bond and van der Waals’ forces and so on.So,it is a tricky task to figure out the amount of the strong denaturing agent.
However, as the biotin tag will be used,your avidin / biotin handbook will be extraordinarily useful for me.
So please send it to me.My email address: wwbsj0401@163.com.
Thank you again for your kind help.Best regards.
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