7 replies to this topic

[Amber_c]

Hi Guys!

I am having trouble running my SDS-PAGE gels. I have run a lot in the past, and this problem has only come up recently. The protein appear as "frowny" faces (see picture), however the markers are nice and flat. This keeps happening no matter what I do.

The gel is 14%, with 5% stacking. The protein samples are in PBS + 0.1% Triton X 100, and are running at the right size. It's a comassie stain.

I have made up new Running Buffer as well as new Tris-HCl and checked all the pHs which are in range.

Any suggestions would be greatly appreciated!

Amber.

[casandra]

Amber_c, on 19 January 2011 - 04:15 PM, said:

Hi Guys!

I am having trouble running my SDS-PAGE gels. I have run a lot in the past, and this problem has only come up recently. The protein appear as "frowny" faces (see picture), however the markers are nice and flat. This keeps happening no matter what I do.

The gel is 14%, with 5% stacking. The protein samples are in PBS + 0.1% Triton X 100, and are running at the right size. It's a comassie stain. example comassie.jpg

I have made up new Running Buffer as well as new Tris-HCl and checked all the pHs which are in range.

Any suggestions would be greatly appreciated!

Amber.

Hi Amber...and welcome to the forum. First a disclaimer- I have not run a lot of SDS PAGE gels in my life and I'm only familiar with the miniprotean system ..I'm pretty sure that the other more knowledgeable members here can help you out. Your gel has some 3-D effects right there. Could it be uneven thickness in your gel...ie your plates are more tightly clamped on the top left side (your smallest marker also started to frown). Have you tried using another assembly unit or running unit in case it's a current problem? If you run your marker on the middle lanes, do they remain nice and flat? And if you load less proteins, do you see the same result?

Edited by casandra, 19 January 2011 - 08:43 PM.

[mdfenko]

i suspect your problems arise from the buffer that your samples are in.

salt can cause problems with conductance and heat. it can cause lanes to skew and distort.

triton can defeat sds (you can remove sds from proteins by dialyzing against triton). triton in the sample's buffer can compete with the sds in the loading buffer.

if you require publishable results, change the buffer of your samples by dialysis prior to electrophoresis (you can perform drop dialysis of enough for the gel).

Edited by mdfenko, 20 January 2011 - 08:05 AM.

[Amber_c]

mdfenko, on 20 January 2011 - 08:04 AM, said:

i suspect your problems arise from the buffer that your samples are in.

salt can cause problems with conductance and heat. it can cause lanes to skew and distort.

triton can defeat sds (you can remove sds from proteins by dialyzing against triton). triton in the sample's buffer can compete with the sds in the loading buffer.

if you require publishable results, change the buffer of your samples by dialysis prior to electrophoresis (you can perform drop dialysis of enough for the gel).

Thanks mdfrenko! I have also suspected the sample buffer as I have changed a number of other things to no effect. However the samples are already in PBS + 0.05- 0.25% triton X100 (and 1X Laemmli buffer). Do you think the sds is still having an effect? I will check for a possible protocol for dialysis. Thanks for your reply. I will let you know if I have any luck!

[Amber_c]

[/quote]
Hi Amber...and welcome to the forum. First a disclaimer- I have not run a lot of SDS PAGE gels in my life and I'm only familiar with the miniprotean system ..I'm pretty sure that the other more knowledgeable members here can help you out. Your gel has some 3-D effects right there. Could it be uneven thickness in your gel...ie your plates are more tightly clamped on the top left side (your smallest marker also started to frown). Have you tried using another assembly unit or running unit in case it's a current problem? If you run your marker on the middle lanes, do they remain nice and flat? And if you load less proteins, do you see the same result?
[/quote]

Thanks for the welcome cassandra.

I am also using the miniprotean 3 system, which doesn't really allow you to adjust the clamps. I have tried a different unit and the gels appear the same. If I load the markers anywhere in the gel they look the same (only the smallest band isn't straight). The amount of protein loaded does not seem to affect the frowny result (I initially thought this might be the problem too).

I am tending towards the theory that it is the sample buffer that the proteins are in. I still have to work out how to change it.

Thanks for your reply. Any other suggestions would be greatly appreciated.

Amber.

[kfunk106]

Amber_c, on 23 January 2011 - 07:33 PM, said:

Thanks mdfrenko! I have also suspected the sample buffer as I have changed a number of other things to no effect. However the samples are already in PBS + 0.05- 0.25% triton X100 (and 1X Laemmli buffer). Do you think the sds is still having an effect? I will check for a possible protocol for dialysis. Thanks for your reply. I will let you know if I have any luck!

I would think that PBS would be fine--mostly you want to keep your NaCl under control. You may want to try running it at a lower voltage, this will keep the heat down and generally look better.

[DustyMS]

Just turn it upside down, so at least you're looking at a smile. I use Pre-Cast gels from Protea Biosciences, as well as MW markers, and I find that even if I'm a little off, the lanes still run fast, and straight.

[DaRQsiDe]

If you are not already doing this, try running the gel in a cold room or cold cabinet and use cold buffers and run at a lower current. That used to work for me in the past.