3 replies to this topic

[Matrix]

Hi guys, was wondering if anyone can help me out with this problem I am experiencing in western

I had been experiencing some inconsistencies in western results quality. Sometimes the individual lanes will show up as distinct clear bands, other times they will merge together into one line even at low developing time. I experienced these often with low percentage gel and with proteins of lower molecular weight. Is it possible that I have run the electrophoresis too long and the low percentage gel become constricted over time? I run the samples at RT, 100 – 120 V.

Any suggestions will be greatly appreciated.

[cellcounter]

Matrix, on Aug 4 2009, 01:54 AM, said:

Hi guys, was wondering if anyone can help me out with this problem I am experiencing in western

I had been experiencing some inconsistencies in western results quality. Sometimes the individual lanes will show up as distinct clear bands, other times they will merge together into one line even at low developing time. I experienced these often with low percentage gel and with proteins of lower molecular weight. Is it possible that I have run the electrophoresis too long and the low percentage gel become constricted over time? I run the samples at RT, 100 – 120 V.

Any suggestions will be greatly appreciated.

That always happens with low MW proteins. I think as these proteins run for a longer time, and are small in size, they stop following the lane boundaries (I think because of diffusion).

If this is a really annoying problem,

1. Try running the gel for a short distance only.
2. Run higher % gel.
3. A gradient gel may also help.

Edited by cellcounter, 04 August 2009 - 06:24 AM.

[Matrix]

cellcounter, on Aug 4 2009, 10:23 PM, said:

Matrix, on Aug 4 2009, 01:54 AM, said:

Hi guys, was wondering if anyone can help me out with this problem I am experiencing in western

I had been experiencing some inconsistencies in western results quality. Sometimes the individual lanes will show up as distinct clear bands, other times they will merge together into one line even at low developing time. I experienced these often with low percentage gel and with proteins of lower molecular weight. Is it possible that I have run the electrophoresis too long and the low percentage gel become constricted over time? I run the samples at RT, 100 – 120 V.

Any suggestions will be greatly appreciated.

That always happens with low MW proteins. I think as these proteins run for a longer time, and are small in size, they stop following the lane boundaries (I think because of diffusion).

If this is a really annoying problem,

1. Try running the gel for a short distance only.
2. Run higher % gel.
3. A gradient gel may also help.

Thanks for the tips! will try out these suggestions next time.

[little mouse]

what is the MW you are interested in? You could maybe try tris tricine gels. I found a little more difficult to separate two bands that are close, but the bands are more focused, and you will see better the small molecular weight (<10 kda)