protein concentration of samples in Laemmli buffer - (Nov/20/2008 )
Hi everyone! My first post here but have been looking up useful tips whenever I encounter a problem for a while now.
My question today is regarding the determination of protein concentration in cell lysates in laemmli buffer. I think this has been brought up in the forum but a while ago. I've tried plain acetone precipitation and not sure on those results and now have also tried another method as follows: To 10 µl boiled lysate (in Laemmli sample buffer) add 40µl water + 50µl 50% TCA. Ppt. 10 min. on ice. Spin and wash with 10% TCA. Resuspend pellet in 100 µl 1M NaOH, add 900 µl water and assay 100 µl by Bradford method.
Anyone uses this method? Any comments?
My other problem is not knowing how much protein to expect. Using this method this is the result I got: from 1x106 cells in 50ul laemmli I had 10.22 ug protein...this doesn't seem like a lot and I would expect more from this amount of cells...
Can anyone shed some light on this?
Thanks a million 
C.
My question today is regarding the determination of protein concentration in cell lysates in laemmli buffer. I think this has been brought up in the forum but a while ago. I've tried plain acetone precipitation and not sure on those results and now have also tried another method as follows: To 10 µl boiled lysate (in Laemmli sample buffer) add 40µl water + 50µl 50% TCA. Ppt. 10 min. on ice. Spin and wash with 10% TCA. Resuspend pellet in 100 µl 1M NaOH, add 900 µl water and assay 100 µl by Bradford method.
Anyone uses this method? Any comments?
My other problem is not knowing how much protein to expect. Using this method this is the result I got: from 1x106 cells in 50ul laemmli I had 10.22 ug protein...this doesn't seem like a lot and I would expect more from this amount of cells...
Can anyone shed some light on this?
Thanks a million
C.
you boil your cells in sds-page buffer directly? why not make a lysis buffer to easily measure concentration?
My question today is regarding the determination of protein concentration in cell lysates in laemmli buffer. I think this has been brought up in the forum but a while ago. I've tried plain acetone precipitation and not sure on those results and now have also tried another method as follows: To 10 µl boiled lysate (in Laemmli sample buffer) add 40µl water + 50µl 50% TCA. Ppt. 10 min. on ice. Spin and wash with 10% TCA. Resuspend pellet in 100 µl 1M NaOH, add 900 µl water and assay 100 µl by Bradford method.
Anyone uses this method? Any comments?
My other problem is not knowing how much protein to expect. Using this method this is the result I got: from 1x106 cells in 50ul laemmli I had 10.22 ug protein...this doesn't seem like a lot and I would expect more from this amount of cells...
Can anyone shed some light on this?
Thanks a million
C.
You can do a dot blot method for determining protein concentration of samples lysed in sample buffer. I normally use protein (BSA) standards and the samples, and blot 1 ul onto a dry nitrocellulose sheet and stain with amido black. Wash off the stain with 50% methanol til the background is clear. This is just visual comparison but works well for western blot and loading control comes out good too. I hope this helps.
Tulip