protein expresses using pGEX4T-3 but not being purified? - trapped in GST (Jun/02/2008 )
Hi to All!
I am using GST tagged pGEX4T-3 vectr. cloned it with rat parotid secretory protein( 23kDa) , induced IPTG induction 1mM 2 hours, at O.D= .8, pellet ws stored at -80,
most of the protein was seen in inclusion bodies, so to solubilise the protein .25% sarkosyl was added along wth PBS buffer, 1mMDTT, 1mM EDTA, protease inhibitor, pellet ws resuspended and sonicated. 4 cycles, 10 secs, speed 4.
protein ws seen in the soluble form.(did western blot as well as coomassie stainin)
So later on ,used the same pellet frm previous expt (stored at -80), added pbs buffer, sarkosyl, dtt, edta, sonicated, thn added Triton X-100( diff % 0, 0.5, 1,2,3,4), added glutathione slurry50%(v/v), incubated for 15mins at 4 degree celcius, centifuged at 1000* g for 10 secs, got matrix and supernatant(ran the sup on gel and cud see very light band), to the matrix pbs buffer wth protease inhibitor were added and washed twice, to the final matrix 4X sds page loading buffer added, kept at 100 degree celcius for 5 mins, loaded the supernatant, n ot nthng on the gel. 
im all confused wht to do....
the whole experiment ws dne on small scale using GE glutathione sepahoose 4 B slurry!
I am using GST tagged pGEX4T-3 vectr. cloned it with rat parotid secretory protein( 23kDa) , induced IPTG induction 1mM 2 hours, at O.D= .8, pellet ws stored at -80,
most of the protein was seen in inclusion bodies, so to solubilise the protein .25% sarkosyl was added along wth PBS buffer, 1mMDTT, 1mM EDTA, protease inhibitor, pellet ws resuspended and sonicated. 4 cycles, 10 secs, speed 4.
protein ws seen in the soluble form.(did western blot as well as coomassie stainin)
So later on ,used the same pellet frm previous expt (stored at -80), added pbs buffer, sarkosyl, dtt, edta, sonicated, thn added Triton X-100( diff % 0, 0.5, 1,2,3,4), added glutathione slurry50%(v/v), incubated for 15mins at 4 degree celcius, centifuged at 1000* g for 10 secs, got matrix and supernatant(ran the sup on gel and cud see very light band), to the matrix pbs buffer wth protease inhibitor were added and washed twice, to the final matrix 4X sds page loading buffer added, kept at 100 degree celcius for 5 mins, loaded the supernatant, n ot nthng on the gel.
im all confused wht to do....
the whole experiment ws dne on small scale using GE glutathione sepahoose 4 B slurry!
Did you try different conditions for expression, like reduced temperature, lower IPTG etc?
hi
ya i did try lower temp also, 24 degree celcius as well as 10 degree celcius but it was best expressed at 37 degree celcius. as far as iptg conc ..i havnt tried lower than 1mM, do u think lower conc can make a difference?
ya i did try lower temp also, 24 degree celcius as well as 10 degree celcius but it was best expressed at 37 degree celcius. as far as iptg conc ..i havnt tried lower than 1mM, do u think lower conc can make a difference?
I presume the protein was still in the inclusion bodies at the lower temps. If not, there is a rule of thumb that says if you drop the temp by 7 degrees, you need to double to induction time. That should fix the problem of lower yield.
Lower IPTG can reduce the level of induction (anecdotal).
An alternative protocol is to solubilise the IBs in urea, then refold by rapid dilution into buffer. This is then applied to the GSH column. Further details, as well as general tips on refolding denatured proteins can be found at the ReFold database, http://refold.med.monash.edu.au
Hi...
iptg induction at 24 degree celcius was done for 4 hours....and 4 degree celcius iptg induction was done overnite...but protein was expressed best at 37 degree celcius!
im gonna increase the volume of protein( that is made to bind wth gst column) , in the ge catalog, its said tht for 1ml culture volume, tke 10 microlitre of glutathione slurry 50%(v/v) ,is it rite?
should i do the whole experiment of binding at 4 degree celcius or at room temp, ive read smewhr whr they say tht the whole expt shub be carried out at 4 degree celcius........im confused!
one more thing..to extract protein from inclusion bodies ive been adding sarkosyl, thts wrking well, i compared the gel of bth inclusion bodies and the soluble protein!
iptg induction at 24 degree celcius was done for 4 hours....and 4 degree celcius iptg induction was done overnite...but protein was expressed best at 37 degree celcius!
im gonna increase the volume of protein( that is made to bind wth gst column) , in the ge catalog, its said tht for 1ml culture volume, tke 10 microlitre of glutathione slurry 50%(v/v) ,is it rite?
should i do the whole experiment of binding at 4 degree celcius or at room temp, ive read smewhr whr they say tht the whole expt shub be carried out at 4 degree celcius........im confused!
If you are expressing at 24C, induce for at least 8 hrs. Try an overnight induction at 20C and see what kind of yield you get. A lower yield of soluble protein beats a higher yield of IB any day!
Binding can be done at either 4C or RT, depending on how stable your protein happens to be (but you're right, 4C is often better because of lower protease activity).
BTW, wud U mnd not writng in shrthnd? it's really hrd 2 ndrstnd what th h#%@$ u're trIng 2 say!!!!!!!! There is no limitation on characters in these postings...
ok,
im sorry
for using short forms!
so you are tryning to say that extracting protein from inclusion bodies with the help of sarkosyl is not a good way of getting high yield of protein?
A lower yield of soluble protein beats a higher yield of IB any day!
wht do u mean by this?