Hi there,

1- No (and anyway, ethanol is preferable to methanol nowadays...)

2- Heating up is accelerating both processes but if you prefer a nice staining go for the "slowly but surely" option. If you are in a hurry go for heating! Even if the result is messy you could repeat the staining process later...

3- What do you call the whole process?

Thanks for your answer,

1. But i wonder that can we use ethanol only or ethanol/acetic acid mix solution for fixation?

3. If you may send me your final protocol, i will be glad to you

I have trouble with stainning and destainning steps. I dont know how i stain and destain my gel. Because a variety of methods exist in literature. So I am waiting your urgent reply.

Best regards. 

1- Use the mix where ethanol replaces methanol

3- If you have staining/destaining problem , use freshly prepared solutions and incubate at room temperature :

staining is  at least 30 minutes (up to overnight) with 0.25% Coomassie Blue in 50% Ethanol, 10% Acetic Acid.

destaining is with the staining solution lacking Coomassie blue with repeated washes until the right contrast is reached.

Thank you very much, I will try this way, hope this time it will work. Best regards.

Dear Evren,

Here you will find more tips:

https://www.researchgate.net/post/Home_made_instant_blue_coomassie_staining_recipe

Regards - Jan

Years ago people were putting ripped up foam rubber into their destain or recirculating buffer through activated charcoal. Its the destain with takes a long time usually. A short hand assessment is that if many different protocols exist to do the exact same thing, there is likely no "best" one, just the one you end up disliking the least.  

The protocol you used may depend on the type of gel you used and the staining method you chose. I assume you are talking about regular Tris-glycine gel and using coomassie blue staining, then here is the protocol I used from manufacturer:

https://tools.lifetechnologies.com/content/sfs/manuals/electrophoresisguide_man.pdf

See page 38. One additional trick is that I used some twisted Kimwipe tissue to absorb the stains during the destaining which would slightly speed up the destain process. Don't let the tissue touch your gel during destaining as this will lead to "blank spot" on the gel. 

I do use microwave stain/destain and usually get at least equally good quality of gel image as the slow protocol. But I'll adjust the timing (~1hr for staining and O/N for destaining if time allows) to improve the quality especially for low abundance bands. I don't use a fix step. I think the fix step might be more critical for slow staining so as to prevent protein band from gradually smearing out during the long process. 

Thank you very much for your all helps and answers. We found the problem, our CBB wasn't eligible to use, (broken). I also had new techniques and tricks all of your answers thank you very much. 

Best regards.