3 replies to this topic

[Tai]

Bromophenol blue

pH =3.0 - 4.6; color change = yellow to blue-violet

After i boiling my loading sample, it was looked like green-yellow. Probably the pH was between 3 - 3.5 I guess.

What could be the reason on my Laemmli buffer ? to make more acidic to my loading sample?
Does low pH could be the problem like on my images?

my laemmli buffer

Quote

Laemmli buffer for 10 mL

dd H2O - 3.29mL
0.5 M Tris-HCl pH 6.8 - 1.25 mL
Glycerol - 1mL
10% SDS - 2 mL
1% Bromphenolblue - 0,5mL
0.5 M EDTA - 0.005 mL
(Roche complete protease inhibitor 1 tablet and dissolve in water 2mL) - 1 mL
50 mM PMSF - 0.4mL
2-Mercap. 0.5mL

if you see on the picture, it look like spot
It was run on NuPage invitrogel 4-12% 1mm and MOPS running buffer from invitrogen as well.

Attached Thumbnails

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Edited by Tai, 24 October 2012 - 01:13 AM.

[bob1]

The green colour is definitely a pH change, I suspect that either your tris buffer is incorrectly prepared or there is something wrong with the lysis buffer.

The "spot" bands in your western are probably caused by incorrect salt content in the sample, but could also be from the gels being a bit degraded (I've seen it before with Nupage).

[Tai]

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.

Quote

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.

Quote

(Roche complete protease inhibitor 1 tablet and dissolve in water 2mL ) - 1 mL
50 mM PMSF - 0.4mL
2-Mercap. 0.5mL

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.

[bob1]

How are you assessing the protein concentration if you have lysed them directly in Laemmli buffer?

Your laemmli recipe is a 5 x recipe - this would account for many of the problems you are having, as you will be having to load a much higher volume of sample with the MCF10a, the salt content will be much bigger than in other samples...