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What is the best way to prepare pichia pastoris whole cell lysates...
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Mechael Kanovsky
What is the best way to prepare pichia pastoris whole cell lysates...
HI - generally speaking if you are looking at a transmembrane protein, I would advise against boiling your sample prior to SDS-PAGE, as TM proteins tend to aggregate in the presence of SDS. As results instead of having a nice band on your gel you might end up with a smear. I generally prepare my own 2X Laemmli sample buffer supplemented with 8M urea and 2M thiourea. Denature at 25C for 30 min. For very difficult TM proteins, providing you cast your own gels, you might want to add urea to your slurry, 4M usually does the trick. As per sample preparation goes, there are a few options, mainly depending on the scale of your expression. 1) incubated the cells with lithium acetate followed by NaOH - the system works really well for soluble proteins. I tested it for TM proteins as well and 90% of the cases it works well.The lysate is quite viscous so to shear genomic DNA pass through a syringe a few times. 2) Check the EasySelect Pichia manual from LifeTechnologies/Invitrogen for small scale mechanical disruption with glass beads in breaking buffer. This also worked for me, however, I tend to load the full lysate withouth centrifugating it first. 3) Spheroplasting - the usual 1.4M sorbitol buffer in the presence of Lyticase or Zymolyase followed by Dounce Homogeniser 4) Beadbeater, a little messier but if you work with liters worth of Pichia colture its probably the way to go OR 5) Dear old French Press.. Hope it helps. Let me know how it goes. Best. Andrea
HI - generally speaking if you are looking at a transmembrane protein, I would advise against boiling your sample prior to SDS-PAGE, as TM proteins tend to aggregate in the presence of SDS. As results instead of having a nice band on your gel you might end up with a smear. I generally prepare my own 2X Laemmli sample buffer supplemented with 8M urea and 2M thiourea. Denature at 25C for 30 min. For very difficult TM proteins, providing you cast your own gels, you might want to add urea to your slurry, 4M usually does the trick. As per sample preparation goes, there are a few options, mainly depending on the scale of your expression. 1) incubated the cells with lithium acetate followed by NaOH - the system works really well for soluble proteins. I tested it for TM proteins as well and 90% of the cases it works well.The lysate is quite viscous so to shear genomic DNA pass through a syringe a few times. 2) Check the EasySelect Pichia manual from LifeTechnologies/Invitrogen for small scale mechanical disruption with glass beads in breaking buffer. This also worked for me, however, I tend to load the full lysate withouth centrifugating it first. 3) Spheroplasting - the usual 1.4M sorbitol buffer in the presence of Lyticase or Zymolyase followed by Dounce Homogeniser 4) Beadbeater, a little messier but if you work with liters worth of Pichia colture its probably the way to go OR 5) Dear old French Press.. Hope it helps. Let me know how it goes. Best. Andrea
Hi Girishkumar, Yes, the shift in the apparent molecular weight after SDS-PAGE is also normal for transmembrane proteins and yes most likely is due to the partial denaturation you obtain without boiling the sample. Generally TM tend to have an apparent molecular weight as per SDS-PAGE goes, that is smaller or bigger than the theoretical one based on amino acid composition... Yes a western is the way to go.. All the best. Regards. Andrea
Hi Girishkumar, Yes, the shift in the apparent molecular weight after SDS-PAGE is also normal for transmembrane proteins and yes most likely is due to the partial denaturation you obtain without boiling the sample. Generally TM tend to have an apparent molecular weight as per SDS-PAGE goes, that is smaller or bigger than the theoretical one based on amino acid composition... Yes a western is the way to go.. All the best. Regards. Andrea
We usually spin down the cells and resuspend the pellet in sufficient amount of 1x Laemmli buffer, boil it and sonicate (only a few seconds, just to get rid of nucleic acids). Then you can load on a gel.. This is just for checking the expression, making cell lysate for purification is a bit different. Sometimes the sonication in PBS is sufficient, but since you have a membrane protein, it can remain in a pellet after sonication and centrifugation. We resuspend the pellet in buffer with 8M urea and some detergent (2% Triton X-100, 0,1% SDS), then mix together with a sonicate and use for isolation
We usually spin down the cells and resuspend the pellet in sufficient amount of 1x Laemmli buffer, boil it and sonicate (only a few seconds, just to get rid of nucleic acids). Then you can load on a gel.. This is just for checking the expression, making cell lysate for purification is a bit different. Sometimes the sonication in PBS is sufficient, but since you have a membrane protein, it can remain in a pellet after sonication and centrifugation. We resuspend the pellet in buffer with 8M urea and some detergent (2% Triton X-100, 0,1% SDS), then mix together with a sonicate and use for isolation
Hi, Thank you all for replies. It seems that the membrane protein I am working on is not as problematic as expected. I did the very basic things and it gave me very clear band. I lysed the cells using sonication in a sonication buffer containing PMSF and DTT. I did not boil the sample, but I incubated it with SDS loading buffer and BME at 37 degree Celsius. Thats all but there was gel shifting because of incomplete denaturation I guess. I am going to do a western to confirm it. Otherwise, my gel look excellent with minimal background Once again, thank you all for your suggestions.
Hi, Thank you all for replies. It seems that the membrane protein I am working on is not as problematic as expected. I did the very basic things and it gave me very clear band. I lysed the cells using sonication in a sonication buffer containing PMSF and DTT. I did not boil the sample, but I incubated it with SDS loading buffer and BME at 37 degree Celsius. Thats all but there was gel shifting because of incomplete denaturation I guess. I am going to do a western to confirm it. Otherwise, my gel look excellent with minimal background Once again, thank you all for your suggestions.
Girishkumar K. Kumaran .. Hi, can you please provide the sonication parameters used by you and the lysis buffer composition. I am struggling with obtaining cell lysis samples from pichia for SDS.
Girishkumar K. Kumaran .. Hi, can you please provide the sonication parameters used by you and the lysis buffer composition. I am struggling with obtaining cell lysis samples from pichia for SDS.
HI - generally speaking if you are looking at a transmembrane protein, I would advise against boiling your sample prior to SDS-PAGE, as TM proteins tend to aggregate in the presence of SDS. As results instead of having a nice band on your gel you might end up with a smear. I generally prepare my own 2X Laemmli sample buffer supplemented with 8M urea and 2M thiourea. Denature at 25C for 30 min. For very difficult TM proteins, providing you cast your own gels, you might want to add urea to your slurry, 4M usually does the trick.
As per sample preparation goes, there are a few options, mainly depending on the scale of your expression.
1) incubated the cells with lithium acetate followed by NaOH - the system works really well for soluble proteins. I tested it for TM proteins as well and 90% of the cases it works well.The lysate is quite viscous so to shear genomic DNA pass through a syringe a few times.
2) Check the EasySelect Pichia manual from LifeTechnologies/Invitrogen for small scale mechanical disruption with glass beads in breaking buffer. This also worked for me, however, I tend to load the full lysate withouth centrifugating it first.
3) Spheroplasting - the usual 1.4M sorbitol buffer in the presence of Lyticase or Zymolyase followed by Dounce Homogeniser
4) Beadbeater, a little messier but if you work with liters worth of Pichia colture its probably the way to go
OR
5) Dear old French Press..
Hope it helps. Let me know how it goes.
Best.
Andrea
HI - generally speaking if you are looking at a transmembrane protein, I would advise against boiling your sample prior to SDS-PAGE, as TM proteins tend to aggregate in the presence of SDS. As results instead of having a nice band on your gel you might end up with a smear. I generally prepare my own 2X Laemmli sample buffer supplemented with 8M urea and 2M thiourea. Denature at 25C for 30 min. For very difficult TM proteins, providing you cast your own gels, you might want to add urea to your slurry, 4M usually does the trick.
As per sample preparation goes, there are a few options, mainly depending on the scale of your expression.
1) incubated the cells with lithium acetate followed by NaOH - the system works really well for soluble proteins. I tested it for TM proteins as well and 90% of the cases it works well.The lysate is quite viscous so to shear genomic DNA pass through a syringe a few times.
2) Check the EasySelect Pichia manual from LifeTechnologies/Invitrogen for small scale mechanical disruption with glass beads in breaking buffer. This also worked for me, however, I tend to load the full lysate withouth centrifugating it first.
3) Spheroplasting - the usual 1.4M sorbitol buffer in the presence of Lyticase or Zymolyase followed by Dounce Homogeniser
4) Beadbeater, a little messier but if you work with liters worth of Pichia colture its probably the way to go
OR
5) Dear old French Press..
Hope it helps. Let me know how it goes.
Best.
Andrea
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VOTE
Hi all,
I did the western and we confirmed it.
Girish
Hi all,
I did the western and we confirmed it.
Girish
More
VOTE
Resuspend the cells in preferred buffer and add protease inhibitor then go for sonication.
Resuspend the cells in preferred buffer and add protease inhibitor then go for sonication.
More
VOTE
Hi Girishkumar,
Yes, the shift in the apparent molecular weight after SDS-PAGE is also normal for transmembrane proteins and yes most likely is due to the partial denaturation you obtain without boiling the sample. Generally TM tend to have an apparent molecular weight as per SDS-PAGE goes, that is smaller or bigger than the theoretical one based on amino acid composition... Yes a western is the way to go..
All the best.
Regards.
Andrea
Hi Girishkumar,
Yes, the shift in the apparent molecular weight after SDS-PAGE is also normal for transmembrane proteins and yes most likely is due to the partial denaturation you obtain without boiling the sample. Generally TM tend to have an apparent molecular weight as per SDS-PAGE goes, that is smaller or bigger than the theoretical one based on amino acid composition... Yes a western is the way to go..
All the best.
Regards.
Andrea
More
VOTE
We usually spin down the cells and resuspend the pellet in sufficient amount of 1x Laemmli buffer, boil it and sonicate (only a few seconds, just to get rid of nucleic acids). Then you can load on a gel.. This is just for checking the expression, making cell lysate for purification is a bit different. Sometimes the sonication in PBS is sufficient, but since you have a membrane protein, it can remain in a pellet after sonication and centrifugation. We resuspend the pellet in buffer with 8M urea and some detergent (2% Triton X-100, 0,1% SDS), then mix together with a sonicate and use for isolation
We usually spin down the cells and resuspend the pellet in sufficient amount of 1x Laemmli buffer, boil it and sonicate (only a few seconds, just to get rid of nucleic acids). Then you can load on a gel.. This is just for checking the expression, making cell lysate for purification is a bit different. Sometimes the sonication in PBS is sufficient, but since you have a membrane protein, it can remain in a pellet after sonication and centrifugation. We resuspend the pellet in buffer with 8M urea and some detergent (2% Triton X-100, 0,1% SDS), then mix together with a sonicate and use for isolation
More
VOTE
Hi,
Thank you all for replies. It seems that the membrane protein I am working on is not as problematic as expected. I did the very basic things and it gave me very clear band. I lysed the cells using sonication in a sonication buffer containing PMSF and DTT. I did not boil the sample, but I incubated it with SDS loading buffer and BME at 37 degree Celsius. Thats all but there was gel shifting because of incomplete denaturation I guess. I am going to do a western to confirm it. Otherwise, my gel look excellent with minimal background
Once again, thank you all for your suggestions.
Hi,
Thank you all for replies. It seems that the membrane protein I am working on is not as problematic as expected. I did the very basic things and it gave me very clear band. I lysed the cells using sonication in a sonication buffer containing PMSF and DTT. I did not boil the sample, but I incubated it with SDS loading buffer and BME at 37 degree Celsius. Thats all but there was gel shifting because of incomplete denaturation I guess. I am going to do a western to confirm it. Otherwise, my gel look excellent with minimal background
Once again, thank you all for your suggestions.
More
VOTE
Girishkumar K. Kumaran ..
Hi, can you please provide the sonication parameters used by you and the lysis buffer composition. I am struggling with obtaining cell lysis samples from pichia for SDS.
Girishkumar K. Kumaran ..
Hi, can you please provide the sonication parameters used by you and the lysis buffer composition. I am struggling with obtaining cell lysis samples from pichia for SDS.
More
VOTE
Hi Girish,
Glad to hear. This paper is slightly dated but gives a good insight into membrane proteins electrophotetic behaviour.
Kind regards.
Andrea
Hi Girish,
Glad to hear. This paper is slightly dated but gives a good insight into membrane proteins electrophotetic behaviour.
Kind regards.
Andrea
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VOTE
There are simple ways for lysis of pichia to prepare cell lysates for SDS-PAGE. One can also use commercially available lysis buffer (see the link below).
Link: http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/cell-lysis/yeast-cell-lysis.html
There are simple ways for lysis of pichia to prepare cell lysates for SDS-PAGE. One can also use commercially available lysis buffer (see the link below).
Link: http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/cell-lysis/yeast-cell-lysis.html
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