cell lysis buffer - (Apr/29/2008 )
Hello,
I am trying to make a lysate of ex vivo isolated enterocytes for SDS-PAGE. I would like to detect 1 cytosolic and 1 transmembrane protein. Therefore I used a Tris-lysis buffer with 1% tritonX100 and protease inhibitors.
I do not know what to expect since it was the first time that I followed this protocol. My cell lysates are however not clear. Does this mean that cells are not lysed?
I added 1ml of lysis buffer to 100-200 mg of cells. I did BCA and measured about 1-2 mg of protein per sample.
Is this to be expected or is it rather low?
Should I centrifuge my sample and only use the supernatans?
Is it true that I should not boil the samples in SDS-buffer for the transmembrane protein?
For now, I froze my samples in lysis buffer at -20 since I thought that this would maybe cause more cell lysis.
Thank you for answering these questions.
-mich-
QUOTE (mich @ Apr 29 2008, 10:41 PM)
Hello,
I am trying to make a lysate of ex vivo isolated enterocytes for SDS-PAGE. I would like to detect 1 cytosolic and 1 transmembrane protein. Therefore I used a Tris-lysis buffer with 1% tritonX100 and protease inhibitors.
I do not know what to expect since it was the first time that I followed this protocol. My cell lysates are however not clear. Does this mean that cells are not lysed?
I added 1ml of lysis buffer to 100-200 mg of cells. I did BCA and measured about 1-2 mg of protein per sample.
Is this to be expected or is it rather low?
Should I centrifuge my sample and only use the supernatans?
Is it true that I should not boil the samples in SDS-buffer for the transmembrane protein?
For now, I froze my samples in lysis buffer at -20 since I thought that this would maybe cause more cell lysis.
Thank you for answering these questions.
I am trying to make a lysate of ex vivo isolated enterocytes for SDS-PAGE. I would like to detect 1 cytosolic and 1 transmembrane protein. Therefore I used a Tris-lysis buffer with 1% tritonX100 and protease inhibitors.
I do not know what to expect since it was the first time that I followed this protocol. My cell lysates are however not clear. Does this mean that cells are not lysed?
I added 1ml of lysis buffer to 100-200 mg of cells. I did BCA and measured about 1-2 mg of protein per sample.
Is this to be expected or is it rather low?
Should I centrifuge my sample and only use the supernatans?
Is it true that I should not boil the samples in SDS-buffer for the transmembrane protein?
For now, I froze my samples in lysis buffer at -20 since I thought that this would maybe cause more cell lysis.
Thank you for answering these questions.
Cloudy super may be due to TritonX100 addition.
Do you add sucrose to lysis buffer ?
It will be good enough to sure complete lysis - to sonicate your samples / If it is not possible thaw/freeze procedure is good enough also for mammalian cells lysis ( freeze with liquid nitrogen and then 37 C water bath - several times)
Also add DNAseI ( 1mg\ml) to avoid DNA smear durind SDS PAGE
-circlepoint-
Thx for the tip. I added glycerol to my lysis buffer but no sucrose.
What about the protease inhibitors during thaw-freeze cycles. Will they still be active? Or is that no problem?
QUOTE (circlepoint @ Apr 30 2008, 08:58 AM)
QUOTE (mich @ Apr 29 2008, 10:41 PM)
Hello,
I am trying to make a lysate of ex vivo isolated enterocytes for SDS-PAGE. I would like to detect 1 cytosolic and 1 transmembrane protein. Therefore I used a Tris-lysis buffer with 1% tritonX100 and protease inhibitors.
I do not know what to expect since it was the first time that I followed this protocol. My cell lysates are however not clear. Does this mean that cells are not lysed?
I added 1ml of lysis buffer to 100-200 mg of cells. I did BCA and measured about 1-2 mg of protein per sample.
Is this to be expected or is it rather low?
Should I centrifuge my sample and only use the supernatans?
Is it true that I should not boil the samples in SDS-buffer for the transmembrane protein?
For now, I froze my samples in lysis buffer at -20 since I thought that this would maybe cause more cell lysis.
Thank you for answering these questions.
I am trying to make a lysate of ex vivo isolated enterocytes for SDS-PAGE. I would like to detect 1 cytosolic and 1 transmembrane protein. Therefore I used a Tris-lysis buffer with 1% tritonX100 and protease inhibitors.
I do not know what to expect since it was the first time that I followed this protocol. My cell lysates are however not clear. Does this mean that cells are not lysed?
I added 1ml of lysis buffer to 100-200 mg of cells. I did BCA and measured about 1-2 mg of protein per sample.
Is this to be expected or is it rather low?
Should I centrifuge my sample and only use the supernatans?
Is it true that I should not boil the samples in SDS-buffer for the transmembrane protein?
For now, I froze my samples in lysis buffer at -20 since I thought that this would maybe cause more cell lysis.
Thank you for answering these questions.
Cloudy super may be due to TritonX100 addition.
Do you add sucrose to lysis buffer ?
It will be good enough to sure complete lysis - to sonicate your samples / If it is not possible thaw/freeze procedure is good enough also for mammalian cells lysis ( freeze with liquid nitrogen and then 37 C water bath - several times)
Also add DNAseI ( 1mg\ml) to avoid DNA smear durind SDS PAGE
-mich-
QUOTE (mich @ Apr 30 2008, 01:05 AM)
Thx for the tip. I added glycerol to my lysis buffer but no sucrose.
What about the protease inhibitors during thaw-freeze cycles. Will they still be active? Or is that no problem?
Cloudy super may be due to TritonX100 addition.
Do you add sucrose to lysis buffer ?
It will be good enough to sure complete lysis - to sonicate your samples / If it is not possible thaw/freeze procedure is good enough also for mammalian cells lysis ( freeze with liquid nitrogen and then 37 C water bath - several times)
Also add DNAseI ( 1mg\ml) to avoid DNA smear durind SDS PAGE
What about the protease inhibitors during thaw-freeze cycles. Will they still be active? Or is that no problem?
QUOTE (circlepoint @ Apr 30 2008, 08:58 AM)
QUOTE (mich @ Apr 29 2008, 10:41 PM)
Hello,
I am trying to make a lysate of ex vivo isolated enterocytes for SDS-PAGE. I would like to detect 1 cytosolic and 1 transmembrane protein. Therefore I used a Tris-lysis buffer with 1% tritonX100 and protease inhibitors.
I do not know what to expect since it was the first time that I followed this protocol. My cell lysates are however not clear. Does this mean that cells are not lysed?
I added 1ml of lysis buffer to 100-200 mg of cells. I did BCA and measured about 1-2 mg of protein per sample.
Is this to be expected or is it rather low?
Should I centrifuge my sample and only use the supernatans?
Is it true that I should not boil the samples in SDS-buffer for the transmembrane protein?
For now, I froze my samples in lysis buffer at -20 since I thought that this would maybe cause more cell lysis.
Thank you for answering these questions.
I am trying to make a lysate of ex vivo isolated enterocytes for SDS-PAGE. I would like to detect 1 cytosolic and 1 transmembrane protein. Therefore I used a Tris-lysis buffer with 1% tritonX100 and protease inhibitors.
I do not know what to expect since it was the first time that I followed this protocol. My cell lysates are however not clear. Does this mean that cells are not lysed?
I added 1ml of lysis buffer to 100-200 mg of cells. I did BCA and measured about 1-2 mg of protein per sample.
Is this to be expected or is it rather low?
Should I centrifuge my sample and only use the supernatans?
Is it true that I should not boil the samples in SDS-buffer for the transmembrane protein?
For now, I froze my samples in lysis buffer at -20 since I thought that this would maybe cause more cell lysis.
Thank you for answering these questions.
Cloudy super may be due to TritonX100 addition.
Do you add sucrose to lysis buffer ?
It will be good enough to sure complete lysis - to sonicate your samples / If it is not possible thaw/freeze procedure is good enough also for mammalian cells lysis ( freeze with liquid nitrogen and then 37 C water bath - several times)
Also add DNAseI ( 1mg\ml) to avoid DNA smear durind SDS PAGE
I think tthat if you will apply lysis sample to SDS-PAGE after this immediately ( in sample buffer ) and don't work with it ( I mean further purification process) so don't worry about this.
-circlepoint-
QUOTE (circlepoint @ Apr 30 2008, 02:58 AM)
Do you add sucrose to lysis buffer ?
Could anybody tell me why we should add sucrose in lysis buffer?
Thanks a lot in advance!
Have a great weekend!
-yuer-