I am producing a recombinant protein in Rosetta, my protein is fused to MBP+his-tags at the amine terminus. I have solubilized it with sarkosyl. I am using IMAC column to purify my protein and thereafter I used TEV protease to remove the fusion protein from my target one. After TEV digestion I ran an SDS-PAGE and I can see the enzyme and the fusion tag (MBP+His) as clear bands while the target protein (IgT) is faint. I want also to mention that running the purified protein before digestion under reduced conditions gave clearer band than the non-reduced condition gave for the same protein.
Dear Haitham
did you observed any precipitate after TEV cutting?
In case, try to centrifuge the sample after tev cleavage at high speed and if you see precipitate in the tube, resuspend it and run in the gel to check it is your protein.
mbp is very soluble and some times can happen that the target protein is soluble just thanks to mbp and after mbp cleavage it precipitare and in the gel you can see only mbp and enzime.
however you use an high amount of enzime and it can in some way also cover the band of your proten. TEV is very efficient is not necessary to use a so high amount. You can certanilly reduce it.
best regards
manuele
Thanks Manuele,
I didn't see any precipitation in the tube may be because I am using only 40 ug of the original purified protein, but my question is could the target protein precipitate in the presence of Sarkosyl?
I am still optimizing the experiment and I agree the enzyme is too much, I will reduce the enzyme next time.
In your opinion, how the enzyme band (27 kDa) could cover the target protein (31 kDa)
Finally, do you have any idea why the purified protein band under reduced condition is much clearer than under the non reduced one?
Do your protein contains cysteines?
is it possibile that with out reducing agent it aggregate and it form high moleciular weight aggregate that are difficult to be seen in the gels?
Of course sarkosyl can prevent protein from precipitation, but at which concentration do you use it? Because cannot be so concentrated if TEV work in it prencence? i'm right?
Dear Haitham
I dont see any significant differences between lane 5 and 6. But I have a little bit problem with analysis of lane 1-3 (Is it equivalent amount of protein in each lane?, the some question for line 3, 5 and 6). You used 40 ug of protein for digestion , but how much of digestion mix did you loaded on the gel? Did you try to do western blot ? In yours gel conditions the enzyme band could not cover the target protein. I see one problem in lane 5 and 6, it is broad smear band between 50-70 kDa, not observed in lane 3.
Jarek
Thanks Jarek,
For lane 5 and 6 we wanted to know if we could digest the protein in absence of DTT. for lane 1-3 amount of protein are equal in all of them, the only difference is that 1,2 lanes are the eluted fraction of the IMAC column in elution buffer run under the reduced and non reduced conditions respectively, while lane 3 is the same amount of protein protein in the digestion buffer without TEV protease (control negative sample for the digestion experiment). and yes it is the same for 3,5,6 lanes and you can see that from the clear bands of MBP.
I loaded 16 ul of the digestion mix.
Yes the smear in lane 5 and 6 lanes may be because of the TEV effect on the 75 kDa r-protein, which is absent in lane 3.
but again do you have any explanation why lane 1 and 2 are not the same although the amount of protein in each of them is the same?
Thanks Srikanth,
I couldn't see any precipitation in the tube after digestion, and do you think that 1% of sarkosyl won't solve this aggregation if it happened.
Hi Haitham
Probably your IgT under normal conditions existed as polimer (tetramer) but in reduced condition it form monomer. You should to see whole gel (wells and stacking gel). On the other hand, the polymer can precipitate without of DTT. I suggest TCA precipitation of the sample before SDS-PAGE.
Jarek
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459821/#!po=16.6667
HI Haitham
1% of sarkosyl is good enough but i know proteins which dissolve only in 1% of sarkosyl with 4M urea or GdHCl.
Jarek
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