Saroj,

What is the lowest molecular weight protein in your ladder is it lower than 35 Kda? sometimes pre stained ladders have very low quantities of lower molecular weight proteins, which appear as faint bands. Also check whether your ladder and protein are being cooked at 95 deg with a reducing agent.  please ensure that your resolving gel has formed correctly all the way through.Please check the strength of your electrophoresis buffer. Try to look at these issues one by one. Hope you solve your problem. All the best

Hi there,

if running tris-glycine SDS/PAGE, your gel is not behaving like a 12% one. Resolution should be efficient down to 10-15kDa. If resolution cut is around 35kDa it means your gel is behaving like a 7-8% one. So first i would check stock solutions and recipe used for preparing the gel. Have a look at the following pictur from Biorad about MWM and gel percentage:

http://www.aesociety.org/areas/images/1d-fig6.jpg

It is certainly baffling. Have you tried reducing the concentration of the separation gel to test if this happens in the same way? I have used as a standard 10% and it worked fine  to separate from 120 to 20 KDa in a 9x6 cm gel (Bio-Rad size). In fact, If I wanted to obtain a better resolution in the range above 100 KDa I only needed to keep the gels running and let anything below 30-40 KDa "escape" to the electropohoresis tank.

Are you buying the gels or preparing them yourself? And are you using them fresh or are they oldish (let's say a month old)? You had a good intuition about the pH, I think, but you cannot check it inside the gel. Typically buffering conditions are not stable on the top and bottom of the gel, since they are open on those sides to air (which means oxygen that can oxydate and acidify or CO2 that solubilizing also acidifies) or whatever preserving buffer. You need high pH in your resolving gel, above 8.5, to keep the SDS ionic and thus the proteins running to the + pole.

On the buffer side, did you also check pH of the buffer on the + electrode side after your run, or after 1 hr of run? In general, do you see a constant amount of current going through the gel from beginning to end or does it decrease markedly (this would tell you the problem might be in the buffer).

Otherwise, I agree with Dominique and Carlos, the acrylamide concentration might be less than you think (if you are casting the gels yourself recalculate, considering that stock acrylamide comes in solution, either 30 or 40%, and bisacrylamide proportions are also different, the less you have, the looser your acrylamide mesh when it polymerizes; if you buy gels, maybe buy a different lot!). An 8% gel normally does not separate below 36 kDa (so, GAPDH runs really in the front, together with the BPB line).

Are u preparing gel by yourself or using ready to use gels. In case you are preparing gel you may check the ingredients in addition to electrophoresis or running buffer. 

• Thank You Prasad Pethe, Dominique Liger, Carlos Ocaña Salceda, Pietro Pilo Boyl  and  Miss Javid. Actually I used to get good result in my previous experiments . Few days ago when my stock solution for Tris 1.8 M of pH 8.8 got finished, I prepared new stock solution of pH 8.8, of same strength but the prestained protein marker as well as BPB line did not migrate below 35 Kda.  Yeah the marker designating  for 28,  17 and 6 kda  got acumulated just below 35 kda marker at same horizontal line with  dark stained .  Off course as mentioned  by Dominique, my sds page gel  is behaving like  10-8%  one. Even I tried doing electrophoresis using prestained marker and loading buffer only, without loading protein sample,  but still the protein marked did not moved below 35 kDa. The recipie  for 12 % sds page I used is:

Water                                       : 10.69 ml

40% Acrylamide-bis(29:1)    ;7.50 ml

1.5 M Tris pH 6.8                    :6.30 ml

10% APS                                :0.25 ml

10% SDS                               :0.25 ml

TEMED                                   : 0.005   ml

Total volume                         : 25 ml

stacking gel: 5%, total volume 5 ml.

 Though I have changed all the ingredient  by fresh one many times, I am not able to resolve my problem. Hope ones again, after changing  all  ingredient used to prepare sds page gel, I will  get ride of my problem. 

Thank you all one again for your valuable comments!

So according to what you tell, it turned bad when preparing gel from new stock of Tris buffer pH8.8 (ie all the other reagents were from the same stocks?)? Then I would reprepare this particular stock first(181.65g of Tris base dissolved in water and adjusted to pH8.8 with HCl then volume adjusted to 1L)

If it is still bad then I would consider changing other stock solutions... Starting with acrylamide.

The very silly question would be : "are you sure the acrylamide stock you use is actually 40% and not 30%?" Because 12% would actually be 9% if 40% was in fact 30%...

Thanks a lot to every one mentioning comment and suggestion. Finally I could seperate the protein band  completely at their specific position during electrophoresis. Actually the pH meter of my lab was not functioning well. So the tris solution I prepared was not of exact desired pH and  I was getting  only partial seperation of  protein during electrophoresis.  After all my attempt, at last I prepared tris solution in another lab, mentained pH using other pH meter and now  i am getting good  result.

Hi Saroj,

Be sure that your stocks are in proper condition. It is better to prepare new SDS gel, 10% APS solution, and running buffer. Also confirm the pH of your TRIS solution in running gel(8.8).

Just a little follow up,

Tris is often not compatible with standard electrodes and you need special tris compatible electrodes.

http://www.bio-rad.com/en-se/faq/268453677/technical-support-faq

hope this helps