how to prepare total cell lysate for Western blot? - (Jul/02/2006 )
hello everybody
i want to know how to prepare total cell lysate from mammilian cells ( adherent and suspension cells)
i mean if i want to start my sample with cells 1x10th6 how to adjust the cell number from the culture
i start by cuture in 5 cm dish and count in trypan blue , if my cells were more than this number its ok i will dilute and adjust but if i have less cells what to do?do i have to centrifuge my cells and then what recount ?????
thankx in advance
Add lysis buffer to the cell and sonicate them on ice.
There is a number lysis buffer......I don't know which one you are going to use.
If cell is less than 1x10^6 cell, then add less lysis buffer.
I hope this may help.
thankx mennie
ok so i will take my whole culture and centrifuge then aspirate the media and resuspend the cells in what PBS ??? about 1 ml??? then count it again and then decide ....
no i amnot asking about the lysis buffer its ok about that , its just the how to get the exact number of 1x10^6 if my cells were not in a confluent state yet.
If you add 1ml of lysis buffer to 1x10^6 cell, then you add 0.8ml of lysis buffer to 0.8x10^6 cell.
Yes, resuspend the cell pellet with PBS, then count.
thankx really
take this pdf its great for anybody who want to know which lysis buffer to use.
http://www.abcam.com/assets/pdf/protocols/WB-beginner.pdf
take this pdf its great for anybody who want to know which lysis buffer to use.
http://www.abcam.com/assets/pdf/protocols/WB-beginner.pdf
Its good information for WB.
hello again
minnie i did use 1 ml lysis buffer to 1x10^6 cell,and i did western yesterday and no protein was founf ,
my professor said that it was too much buffer for the cells
he uses 100 micron lysis buffer for 1x10^6 cell
thankxx
minnie i did use 1 ml lysis buffer to 1x10^6 cell,and i did western yesterday and no protein was founf ,
my professor said that it was too much buffer for the cells
he uses 100 micron lysis buffer for 1x10^6 cell
thankxx
I don't think it is too much: I use 100ul of buffer for 200 000 cells. You could try to quantify your proteins after lysis using Bradford or BCA assays and load not less than 10ug. If you are trying to detect an endogenous protein, be sure it's expressed (and not at too low levels). You could also use an anti-actin or other houskeeping to see that you have enough sample to be detected on your membrane
I use 1 ml lysis buffer to 1X10^7 cells and got cell lysate protein ~ 0.1 to 0.5 mg/ml.
I tried 1 ml lysis buffer to 1X10^8 cells and got cell lysate protein ~ 1.5 to 3 mg/ml.
If you've got a low cell lysate protein concentration after you've measured with Bradford or BCA, just decrease the volume of lysis buffer or increase the cell number.
Hope it works.