Thank you very much for your reply as always !!
Normally, I made stock Laemmi buffer and kept them in-20 C, I just need to have the same batch of laemmli buffer because I run experiment for the whole year to collect my cell samples.
stock Laemmi buffer and kept them in -20 C
dd H2O - 3.29mL
0.5 M Tris-HCl pH 6.8 - 1.25 mL <-- This tris is about more than 2 years old I think
Glycerol - 1mL
10% SDS - 2 mL
1% Bromphenolblue - 0,5mL
0.5 M EDTA - 0.005 mL
when I need to mix with my cell sample, I will add fresh of these inhibitors.
(Roche complete protease inhibitor 1 tablet and dissolve in water 2mL ) - 1 mL
50 mM PMSF - 0.4mL
So far it always work fine with other cells but I just got the new cells line, MCF10A, and its total protein concentration is about 4-6 time higher than other cell line that I have. So, I have to add more laemmli buffer than other cells lines
Other cell line 1x10^6 cells per 300 ul Laemmli
This MCF10A 1x10^6 cells per 600 ul Laemmli <------ Is this could be to make much more salt content in my sample?
If the salt
content in the sample could make the "spot" problem -- Do you mean it was from cells content or from the laemmli buffer?
From the actin.jpg
, if look at the first two lane.
Lane 1. total protein concentration 1.02 ug/ul and I loaded 7.3 ul to get 7.5ug
Lane 2. total protein concentration 0.39 ug/ul and I loaded 19.3 ul to get 7.5ug
Lane 3 is repeated of lane 1.
Other lane also about 0.4 ug/ul of total protein.
PS. I just bought new gel, and it is about 1 month from company
Edited by Tai, 24 October 2012 - 02:20 AM.
I h..ve ne..er finish.. ...ny thi .....