how to deal with protein degradation - (Nov/20/2007 )
Dear all,
Does anyone work with C.elegans’ protein? I try to purify a reconstructed Myosin 9 of C. elegans from Sf9 cells using a FLAG-tag. My problem is, the protein degrades immediately after purification. The elution buffer I used is 20mM Hepes, PH7.4, 30mM KCl, 2mM MgCl, 1mM 2-mercaptoethanol, 4mM EGTA and 50ug/ml. I tried to put some protease inhibitor in the elution buffer, but still didn’t work. Can anyone give any ideas?
Thanks a lot...
Does anyone work with C.elegans’ protein? I try to purify a reconstructed Myosin 9 of C. elegans from Sf9 cells using a FLAG-tag. My problem is, the protein degrades immediately after purification. The elution buffer I used is 20mM Hepes, PH7.4, 30mM KCl, 2mM MgCl, 1mM 2-mercaptoethanol, 4mM EGTA and 50ug/ml. I tried to put some protease inhibitor in the elution buffer, but still didn’t work. Can anyone give any ideas?
Thanks a lot...
50ug/ml what?
add protease inhibitors to the extraction buffer.
are you lysing the flag-tag? if so, then you should find out if the protease for this also lyses the myosin.
myosin is a good substrate for many proteases.
Does anyone work with C.elegans’ protein? I try to purify a reconstructed Myosin 9 of C. elegans from Sf9 cells using a FLAG-tag. My problem is, the protein degrades immediately after purification. The elution buffer I used is 20mM Hepes, PH7.4, 30mM KCl, 2mM MgCl, 1mM 2-mercaptoethanol, 4mM EGTA and 50ug/ml. I tried to put some protease inhibitor in the elution buffer, but still didn’t work. Can anyone give any ideas?
Thanks a lot...
50ug/ml what?
add protease inhibitors to the extraction buffer.
are you lysing the flag-tag? if so, then you should find out if the protease for this also lyses the myosin.
myosin is a good substrate for many proteases.
mdfenko, thank you for answering.
i am sorry. 50ug/ml FLAG-peptide.
yes, i had added protease inhibitors to the lysis buffer.
actually, if I load the sample in a SDS-PAGE gel immediatlly after purification, the band of the homogenate is fine, as well as the bands of the eluted faction. but after few hours later, lets say 2 hours, the bands will degrade into many bands.
so far, i am not lysing the flag-tag.
it seems that the myosin is not stable in the elution buffer. i am thinking of changeing the buffer, but i have to use this buffer for the ATPase assay. I would not like to dialyse the myosin, because the myosin always get precipitation and degradation during the dialysis.
my boss said nomally the myosin can be stable for few days in this buffer. i have no ideas what to do next.
the funny thing is eventhough the myosin degraded into many bands, its activity is good. i don't understand this.
Please help! thanks a million...
most protease inhibitors will have no effect on myosin but not having them around will allow the myosin to be degraded.
as for the greater activity of the degraded myosin: you may be producing heavy meromyosin and subfragment-1. both of these exhibit higher atpase activity than intact myosin.
i also notice that you are using a low kcl concentration in your buffer. the myosins that we worked with required higher kcl to remain in solution (hmm and s-1 do not). (i know, the kcl interferes with sds-page, we learned to live with it and still got good looking gels).
mdfenko, thanks for your quick answer.
and do you think the pH will affect the stability of the myosin 9? because i found that the living pH of C.elegans is pH6.0.
though the C.elegans myosin 9 was expressed in sf9 cells, do you agree that pH7.5 is not so proper?
thanks
and do you think the pH will affect the stability of the myosin 9? because i found that the living pH of C.elegans is pH6.0.
though the C.elegans myosin 9 was expressed in sf9 cells, do you agree that pH7.5 is not so proper?
thanks
i can't speak about c. elegans myosin in particular, because i never worked with it, but we stored all of our myosins at pH 7.5.
i can't speak about c. elegans myosin in particular, because i never worked with it, but we stored all of our myosins at pH 7.5.
[/quote]
do you mean even myosins from other organisms not just human or rat?
by the way, what kind of buffer do you use for ATPase assay? i found different groups usually use different kinds of buffer for the assay. does it matter much?
by the way, what kind of buffer do you use for ATPase assay? i found different groups usually use different kinds of buffer for the assay. does it matter much?
i've worked with myosins from chicken, rabbit, bovine, dog and human and from various tissues. we stored all of them at pH 7.5.
you want to use buffers that don't bind up your cofactors (ie-divalent cations). we use tris, imidazole, acetate, borate depending on the pH at which we wish to assay.
you want to use buffers that don't bind up your cofactors (ie-divalent cations). we use tris, imidazole, acetate, borate depending on the pH at which we wish to assay.
mdfenko, thank you so much for all the answer. wish you a good day ;-)
hello, mdfenko, i am sorry to trucble you again.
i tried the method you suggested for storing the myosins and it works. i am really happy about that. thanks a lot 
however, problem just comes one after another.
so far i am trying the F-actin cosedimentation assay. briefly, myosin was mix with 4uM F-actin in 20mM Hepes, pH7.4; 50mM KCl; 2mM MgCl; 1mM EGTA and 1mM DTT. the samples were incubated at 4C (or room temperature) for 30min, then centrifuged 110,000g for 30min.
and the problem is, i always found the myosin-actin binding was weak. nomal speaking, there should be about 80-90% of myosin binds to F-actin. but in my case, only 40-50%. it did bind well one or two times. but it didn't work most of the time. someone suggests this is nomal. but i wonder that is true. do you ever have any experience of doing the cosedimentation assay yourself? if so, would you please give me some suggestions? i alwasy think maybe you can see the points i didn't see. i can get no ideas from reading the literatures. all the menthods they used neally the same.
looking forward to hearing from you. thanks a lot.