It was not destained initially. For that reason you got extra bands after 48 h. My suggestion is to use dH2O as destaining solution instead off the chemicals as you mentioned. Change the water periodically (every 10 min) you will get clear bands with white background. I use to do like this and i got good gel images.  

It was not destained initially. For that reason you got extra bands after 48 h. My suggestion is to use dH2O as destaining solution instead off the chemicals as you mentioned. Change the water periodically (every 10 min) you will get clear bands with white background. I use to do like this and i got good gel images.  

Hi

I use  a fast technique to destain SDS-PAGE and it very efficient. I use it for after comassie staining. After SDS-PAGE, put the gel in water and boil it in the microwave. you have to stop immediately  when the gel has started boiling. repeat the procedure 3 times, and then put your gel in coomassie, boil it once. remove the coomassie et then put water and boil it again. So, in less then 10 minutes your gel is destained and you have clear bands, and you can just put water for some hours to get read of the extra staining.

Best

C. Galiger

we have observed clear bands when destained with water periodically, thanks shiyam and lorraine for the important  suggestions. i want to know that instead of KimWipe normal wipes could be used to absorb leftover or not?

Just use plain DI water, change the water multiple times and de-stain over night, sometimes even for another day, if the background is high. I have never had issues with water only and I get good results. Good luck!

thanks srgjan and nathalie. i faced a issue i have got bands two times but on third time i didnt get any band instead i got a smear. what could be the cause? i thought of sample buffer.. the same i have used 4-5 times, how many times a sample buffer could be used?

thanks a lot Srgjan,

i make gels by my own with Conventional method. we are not using precast gels. i am doing aggregation study of proteins, in 2 experiments i got bands at same kDa weight but after that i didnt get the band and got smear. 5X SDS sample buffer i make and add b-mercaptoethanol at that time only. i will follow your advice to add reducing reagent on the day of my expermient.

Hi Neelakant

Concerning your question about the possibility to use the buffer several time, I think you should consider the fact that the running buffer it is usually a Tris-Glicine buffer, whereas the gel it is usually prepared in Tris-HCl. As you maybe know very well, the presence of this couple of anion (Cl- and Glicinate) is at the very base of the stacking process during SDS-PAGE. Any time you run a gel HCl goes into the anod container, whereas some og the glicinate remain in the gel while migrating. The net result of this is that at each run you deprivate the running buffer of Glicinate and enrich it in Cl-. Of course, if you use again the running buffer, this is going to affect the sharpness of the bands. 

30% methanol and 10% glacial acetic acid is the best recipe for destining buffer for SDS page for better band clarity visualize.