Even I have started working on Pichia pastoris and faced the same problem. Will surely try the 'heat-denaturation' technique to check the expression of my desired protein.
Even I have started working on Pichia pastoris and faced the same problem. Will surely try the 'heat-denaturation' technique to check the expression of my desired protein.
Rajput Bhagchand After my experience with my problem now i can able to see my protein expression. If you are facing the same problem my suggestion would be dont go for cloning repeatedly now and try to check your protein expression for the confirmed clones by loading your sample on SDS_PAGE followed by silver staining. May be your protein is expressing at low level and also try to confirm your protein expression using ELISA. In shake flask experiments initially i was getting around 2-4 mg per litre of protein for which i checked expression through silver staining. One more approach you can try is heat denaturation where you heat your supernatant(100ml) at 95 degree for about 2hrs and centrifuge complete supernatant at high speed after which you will have small precipitate, dissolve this precipitate in 40-50 ul of buffer and load around 30ul on to SDS-PAGE followed by coomassie staining. with this approach i got to see my protein expression.
Rajput Bhagchand After my experience with my problem now i can able to see my protein expression. If you are facing the same problem my suggestion would be dont go for cloning repeatedly now and try to check your protein expression for the confirmed clones by loading your sample on SDS_PAGE followed by silver staining. May be your protein is expressing at low level and also try to confirm your protein expression using ELISA. In shake flask experiments initially i was getting around 2-4 mg per litre of protein for which i checked expression through silver staining. One more approach you can try is heat denaturation where you heat your supernatant(100ml) at 95 degree for about 2hrs and centrifuge complete supernatant at high speed after which you will have small precipitate, dissolve this precipitate in 40-50 ul of buffer and load around 30ul on to SDS-PAGE followed by coomassie staining. with this approach i got to see my protein expression.
Have you checked the intracellular fraction? The protein might not make it all the way through the secretory pathway for a number of reasons, so it is important to find out if this is a case of no expression at all, or failure to be secreted.
Have you checked the intracellular fraction? The protein might not make it all the way through the secretory pathway for a number of reasons, so it is important to find out if this is a case of no expression at all, or failure to be secreted.
Now I'm working on Protein expression by using Pichia and I also got this problem. Thank you so much for all of your idea and recommendation. I'll try to check my protein again.
Now I'm working on Protein expression by using Pichia and I also got this problem. Thank you so much for all of your idea and recommendation. I'll try to check my protein again.
Even I have started working on recombinant protein expression in Pichia.. but facing similar issues... I am trying to express my protein extracellularly.. but see no expression. I checked for intracellular expression as well but no bands on SDS..
Even I have started working on recombinant protein expression in Pichia.. but facing similar issues... I am trying to express my protein extracellularly.. but see no expression. I checked for intracellular expression as well but no bands on SDS..
Dear Sir Srikanth Katla , I am facing the same issues as yours. Can i know the size of your protein? the proteins size that I tried to express are 42 kda, 31 kda, 26 kda, 19 kda and 15 kda (they were derived from the same structure ectodomain). I successfully expressed the 42kda protein. Unexpectedly, the other 4 proteins were failed to expressed using the same protocol/procedure.
I did check the pellet in case my protein expressed as insoluble protein (inclusion body) by protein extraction but still negative results. I tried to optimize the protein expression by varying methanol induction at 1%, 2% and 3% v/v . Unfortunately, my protein still could not be secreted / detected by SDS-PAGE either by loading supernatant directly or after concentrating the sample by Acetone Precipitation . My protein supposedly expressed in supernatant.
Will try the 'heat-denaturation' technique to check the expression of my desired protein.
Dear Sir Srikanth Katla , I am facing the same issues as yours. Can i know the size of your protein? the proteins size that I tried to express are 42 kda, 31 kda, 26 kda, 19 kda and 15 kda (they were derived from the same structure ectodomain). I successfully expressed the 42kda protein. Unexpectedly, the other 4 proteins were failed to expressed using the same protocol/procedure.
I did check the pellet in case my protein expressed as insoluble protein (inclusion body) by protein extraction but still negative results. I tried to optimize the protein expression by varying methanol induction at 1%, 2% and 3% v/v . Unfortunately, my protein still could not be secreted / detected by SDS-PAGE either by loading supernatant directly or after concentrating the sample by Acetone Precipitation . My protein supposedly expressed in supernatant.
Will try the 'heat-denaturation' technique to check the expression of my desired protein.
Dear Srikanth Katla, I think, 1. your secreted protein may be degraded by proteases. PMSF can be useful. 2. In some cases the integrated gene is released from Pichia genome. check the gene integrity again. 3. Bacterial infection and following protease secretion may degrade the proteins. Check the cultured population by gram staining. 4. After the day 4, instantly check the expressed proteins by unreduced SDS-PAGE. do not store the solution in refrigerator. Regards, Kamran
Dear Srikanth Katla, I think, 1. your secreted protein may be degraded by proteases. PMSF can be useful. 2. In some cases the integrated gene is released from Pichia genome. check the gene integrity again. 3. Bacterial infection and following protease secretion may degrade the proteins. Check the cultured population by gram staining. 4. After the day 4, instantly check the expressed proteins by unreduced SDS-PAGE. do not store the solution in refrigerator. Regards, Kamran
Alejandro Martin Sir i have checked the intracellular fraction but i didn't find my protein of interest. is there chances that my protein didnt express at all.if so how can i overcome it. Prior to my expression studies i confirmed integration of gene of interest in pichia genome by colony PCR where i got two bands one corresponds to AOX1(2.2kb) and other my insert(1.1kb) with 5'AOX and 3'AOX primers. Thank You
Alejandro Martin Sir i have checked the intracellular fraction but i didn't find my protein of interest. is there chances that my protein didnt express at all.if so how can i overcome it. Prior to my expression studies i confirmed integration of gene of interest in pichia genome by colony PCR where i got two bands one corresponds to AOX1(2.2kb) and other my insert(1.1kb) with 5'AOX and 3'AOX primers. Thank You
I tried heat denaturation at 95 degree but got all precipitaion in sds-page.
I tried heat denaturation at 95 degree but got all precipitaion in sds-page.
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Even I have started working on Pichia pastoris and faced the same problem. Will surely try the 'heat-denaturation' technique to check the expression of my desired protein.
Even I have started working on Pichia pastoris and faced the same problem. Will surely try the 'heat-denaturation' technique to check the expression of my desired protein.
More
VOTE
Rajput Bhagchand After my experience with my problem now i can able to see my protein expression. If you are facing the same problem my suggestion would be dont go for cloning repeatedly now and try to check your protein expression for the confirmed clones by loading your sample on SDS_PAGE followed by silver staining. May be your protein is expressing at low level and also try to confirm your protein expression using ELISA. In shake flask experiments initially i was getting around 2-4 mg per litre of protein for which i checked expression through silver staining. One more approach you can try is heat denaturation where you heat your supernatant(100ml) at 95 degree for about 2hrs and centrifuge complete supernatant at high speed after which you will have small precipitate, dissolve this precipitate in 40-50 ul of buffer and load around 30ul on to SDS-PAGE followed by coomassie staining. with this approach i got to see my protein expression.
Rajput Bhagchand After my experience with my problem now i can able to see my protein expression. If you are facing the same problem my suggestion would be dont go for cloning repeatedly now and try to check your protein expression for the confirmed clones by loading your sample on SDS_PAGE followed by silver staining. May be your protein is expressing at low level and also try to confirm your protein expression using ELISA. In shake flask experiments initially i was getting around 2-4 mg per litre of protein for which i checked expression through silver staining. One more approach you can try is heat denaturation where you heat your supernatant(100ml) at 95 degree for about 2hrs and centrifuge complete supernatant at high speed after which you will have small precipitate, dissolve this precipitate in 40-50 ul of buffer and load around 30ul on to SDS-PAGE followed by coomassie staining. with this approach i got to see my protein expression.
More
VOTE
Have you checked the intracellular fraction? The protein might not make it all the way through the secretory pathway for a number of reasons, so it is important to find out if this is a case of no expression at all, or failure to be secreted.
Have you checked the intracellular fraction? The protein might not make it all the way through the secretory pathway for a number of reasons, so it is important to find out if this is a case of no expression at all, or failure to be secreted.
More
VOTE
I am also getting same problem still not getting any solution
I am also getting same problem still not getting any solution
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VOTE
Now I'm working on Protein expression by using Pichia and I also got this problem. Thank you so much for all of your idea and recommendation. I'll try to check my protein again.
Now I'm working on Protein expression by using Pichia and I also got this problem. Thank you so much for all of your idea and recommendation. I'll try to check my protein again.
More
VOTE
Even I have started working on recombinant protein expression in Pichia.. but facing similar issues... I am trying to express my protein extracellularly.. but see no expression. I checked for intracellular expression as well but no bands on SDS..
Even I have started working on recombinant protein expression in Pichia.. but facing similar issues... I am trying to express my protein extracellularly.. but see no expression. I checked for intracellular expression as well but no bands on SDS..
More
VOTE
Dear Sir Srikanth Katla ,
I am facing the same issues as yours. Can i know the size of your protein?
the proteins size that I tried to express are 42 kda, 31 kda, 26 kda, 19 kda and 15 kda (they were derived from the same structure ectodomain). I successfully expressed the 42kda protein. Unexpectedly, the other 4 proteins were failed to expressed using the same protocol/procedure.
I did check the pellet in case my protein expressed as insoluble protein (inclusion body) by protein extraction but still negative results. I tried to optimize the protein expression by varying methanol induction at 1%, 2% and 3% v/v . Unfortunately, my protein still could not be secreted / detected by SDS-PAGE either by loading supernatant directly or after concentrating the sample by Acetone Precipitation . My protein supposedly expressed in supernatant.
Will try the 'heat-denaturation' technique to check the expression of my desired protein.
Thanks.
Dear Sir Srikanth Katla ,
I am facing the same issues as yours. Can i know the size of your protein?
the proteins size that I tried to express are 42 kda, 31 kda, 26 kda, 19 kda and 15 kda (they were derived from the same structure ectodomain). I successfully expressed the 42kda protein. Unexpectedly, the other 4 proteins were failed to expressed using the same protocol/procedure.
I did check the pellet in case my protein expressed as insoluble protein (inclusion body) by protein extraction but still negative results. I tried to optimize the protein expression by varying methanol induction at 1%, 2% and 3% v/v . Unfortunately, my protein still could not be secreted / detected by SDS-PAGE either by loading supernatant directly or after concentrating the sample by Acetone Precipitation . My protein supposedly expressed in supernatant.
Will try the 'heat-denaturation' technique to check the expression of my desired protein.
Thanks.
More
VOTE
Dear Srikanth Katla,
I think,
1. your secreted protein may be degraded by proteases. PMSF can be useful.
2. In some cases the integrated gene is released from Pichia genome. check the gene integrity again.
3. Bacterial infection and following protease secretion may degrade the proteins. Check the cultured population by gram staining.
4. After the day 4, instantly check the expressed proteins by unreduced SDS-PAGE. do not store the solution in refrigerator.
Regards,
Kamran
Dear Srikanth Katla,
I think,
1. your secreted protein may be degraded by proteases. PMSF can be useful.
2. In some cases the integrated gene is released from Pichia genome. check the gene integrity again.
3. Bacterial infection and following protease secretion may degrade the proteins. Check the cultured population by gram staining.
4. After the day 4, instantly check the expressed proteins by unreduced SDS-PAGE. do not store the solution in refrigerator.
Regards,
Kamran
More
VOTE
Please, can any colleague support me with a soft copy of Pichia protocols Book ( cat No: G100-01) of invitrogen company?
Please, can any colleague support me with a soft copy of Pichia protocols Book ( cat No: G100-01) of invitrogen company?
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VOTE
Alejandro Martin Sir i have checked the intracellular fraction but i didn't find my protein of interest. is there chances that my protein didnt express at all.if so how can i overcome it. Prior to my expression studies i confirmed integration of gene of interest in pichia genome by colony PCR where i got two bands one corresponds to AOX1(2.2kb) and other my insert(1.1kb) with 5'AOX and 3'AOX primers.
Thank You
Alejandro Martin Sir i have checked the intracellular fraction but i didn't find my protein of interest. is there chances that my protein didnt express at all.if so how can i overcome it. Prior to my expression studies i confirmed integration of gene of interest in pichia genome by colony PCR where i got two bands one corresponds to AOX1(2.2kb) and other my insert(1.1kb) with 5'AOX and 3'AOX primers.
Thank You
More
VOTE