3 replies to this topic

[p3t3r1]

Hi, I ran an induction timecourse a couple days ago and found my protein to be intact and in significant quantities in all induction times used. The gel is shown below. At each induction time period, I take some cells and put them into SDS sample buffer and boil them at 95 C before load them on the gel. My desired protein is 52 kD.  



However, when I do my purification and elute the proteins off the beads, I see two significant degradation bands around 25 kD and I observe co-purification with my protein significant quantities (more than my protein) of DnaK protein, which is a Hsp70 chaperon (this is seen in the gel below as the huge band above my protein band, which is marked with an arrow) In my lysis buffer, I have  1mM PMSF + Roche protease inhibitor tablet and I do all purification steps in the cold room at 4 C. Hence, I cannot think of any reason why my protein would be degraded in such large quantities unless my lysis condition is too harsh. I have the evidence of this harsh lysis condition because the co-purification of DnaK, which according to GE lifesciences (company that made the GST beads) is due to harsh lysis conditions. This got me thinking, is it possible for soniciation to lyse my cells to be too harsh? I soniciate for 3 times @ 10 secs each with 30 secs rest in between. I repeat this for a total of 7 times which means I essentially soniciate 21 times for 10 secs each time. I keep my all my samples on ice during soniciation and cool the probe whenever necessary.

Thanks!

Edited by p3t3r1, 15 August 2009 - 02:30 PM.

[swanny]

As far as I recall, sonication won't degrade proteins, especially given the parameters  you mention. What sonication can do is denature protein, but you have to hit it very hard for that to happen; the effects are mostly thermally derived, and in any case, that would just lead to a reduced total yield of protein, not what you see.

I think the problem lies in your purification. How old are your solutions? What are the components of the lysis buffer? What timeframe does your preparation take? What column are you using? Do you/ how do you regenerate the column? Try with completely fresh reagents and see if that makes any difference.


Oh, and good luck.

[HomeBrew]

The easiest way to check this would be to perform your lysis by another method (chemical, French press) and see if it makes a difference in the amount of degradation products you see.

[p3t3r1]

swanny, on Aug 16 2009, 03:11 PM, said:

As far as I recall, sonication won't degrade proteins, especially given the parameters  you mention. What sonication can do is denature protein, but you have to hit it very hard for that to happen; the effects are mostly thermally derived, and in any case, that would just lead to a reduced total yield of protein, not what you see.

I think the problem lies in your purification. How old are your solutions? What are the components of the lysis buffer? What timeframe does your preparation take? What column are you using? Do you/ how do you regenerate the column? Try with completely fresh reagents and see if that makes any difference.


Oh, and good luck.

Thanks for the reply, my lysis buffer consists of DTT, triton X-100, ATP, MgCl2, lysozyme, PMSF, protease inhibitor tablet, PBS, DNase. I am using packing the column myself by using a reuseable column and pack it with Glutathione Sepharose 4B. I did regenerate my beads/column but that makes little difference since I used fresh ones before and the same results were obtained. My purification timeframe is as follows: lyse the cells in lysis buffer using sonicator, then spin down at 15,000 rpm for 30 min, then take that supernatent and spin again at 18,000 rpm for 30 min (all done at 4 C). Afterwards, in the cold room, I run my supernatent over the column 3 times, in total taking about 30-40 minutes. Then I wash the columns with wash buffer containing DTT, triton-X 3 times, taking 15 minutes each time. (10 mintues for the wash buffer to incubate the beads and then the remaining 5 mintues for the wash buffer to drip out of the column). After wards, I elute using reduced gluthionine (10 mg/ml, pH 8.0) for 5 times and each time taking 11 minutes (10 minutes to incubate with beads, 1 minute to drip).