inclusion bodies? - (Jun/21/2007 )

hi new PhD student here. ive just produced a recombinant protein in ek/lic pet30 in BL21de3plys cells. i used bugbuster to lyse the cells and purified the supernatant. following purification under native and denturing conditions (urea) i get a thick band about two to three times the size of my expected protein size with some VERY minor larger and smaller bands. my question is WHAT IS HAPPENING?
is the protein forming an inclusion body? i thought inclusion bodies would only be found in the pellet following bugbuster treatment. am i wrong? if it isnt what is it and how do i get the protein to the correct size?
any ideas would be great as i am very confused,
thanks
bob

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your protein would be formed in trimer. You can check it by WB.

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thanks for the reply NTH! i'm just waiting for my antibody to come in should be here soon.. so im onto that. i treated with DTT and B-ME should this not break the trimer/dimer up into its respective monomer?

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That would depends on how the polimer is formed. DTT and 2ME only reduce certain bonds such as s-s. If the monomers are linked by other bonds then you will still have dimer/trimer...

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are inclusion bodies only in the pellet of the bugbuster extraction?.any ideas how to break it up into the monomer then?

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you may be able to break it to the monomer with denaturing conditions. along with the b-me (or dtt) you can use sds (with or without heating) or urea.

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Inclusion body are usually insoluble, so you'd expect them in the pellet, yes. It seems to me that you are purifying some other protein than your protein of interest. Urea, BME, DTT and SDS should all be able to break dimer/trimer, unless they are formed by covalent bonds (wich is highly improbable). One last test you can make is to boil your sample before loading it onto a gel. If the band is still there, then it probably is a foreing protein.

What are you using to purify your protein? Any particuliar tag?

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Hello b_c!

Try to make first PAGE with your bacterial cells total lysate. Take a little portion of cell suspension, add water equal volume, sonicate, add BME sample buffer and make PAAG. And estimate expression level of your protein first. And it will be good to make WB of cell lisate too.

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