Not sure of what % to use for gels - (Jan/12/2010 )

I'm trying to do a western blot.
I had my cell lysate samples prepared (Lysate in RIPA buffer along with sample buffer with mercaptoethanol added)

The protein of interest should be about 95kDA.
I ran a gel of 10% sorting gel and 4% of stacking gel.

However, when i loaded the samples and set voltage at 100V, they reached the sorting gel and took forever to run into the sorting gel.
The ladder i added showed that bands for 0-about 50 kda went through fine, but everything else stalled at the top.

I reasoned that my gel was not right.

Should i go with a higher or lower % of sorting gel?
I've thought a lower percentage would work better, but then i've seen some protocols suggesting a higher one.

Is there anything else that could maybe be going wrong?

could you try a gradient gel (eg 5-15%).

if your sample won't migrate into 10% then you can try 7.5% (the old laemmli standard for general purpose gels).

as for how long it took to run through the running (sorting) gel, are you sure that your buffers were made correctly? and that you chose an appropriate current (or voltage) to run the gel?

mdfenko on Jan 12 2010, 05:33 PM said:

The voltage was 100V, but i then tried 150-200 as it was two hours later and the sample had barely moved.
I remade some of the buffers, namely the Tris-HCl (as i'm suspect of the ph being wrong. i use a 8.8 and 6.8 for the sorting and stacking gel, respectively)
I'm also going for a 7.5% gel, to see if that makes any difference.

The range of proteins kept in a 10% gel is from 30 to 250 kda (from what I see with MW markers). Thus, it seems to me that your gels/buffers/running conditions are not right. I frequently use 4 % stacking and 10% separating gels.

Could you provide your buffer and gel recipe ?

madrius1 on Jan 12 2010, 06:25 PM said:

7.5% gel:

4.85mls dH20.
2.5mls of 1.5M tris-hcl ph 8.8
2.5mls acrylamide bis
100ul 10% SDS

50ul of 10% APS
5ul of temed. (Added last)

in a 10% gel, it's 4mls of dH2o and 3.33mls of acrylamide bis, but everything else is the same.


The stacking gel is at 4%:
3.05mls of dH2O
1.25mls of 0.5M Tris-Hcl ph 6.8
675ul of acrylamide bus
50ul of 10% SDS

50ul of 10% APS
5ul of TEMED (added last).

i think that the buffer most likely to be off is the tris-glycine electrode buffer. your sample passes through the stack well enough. it slows down as the electrode buffer catches up (a little simplified).

Agree with mdfenko.