Hi,
I am intent to run Tris/Glycine SDS-PAGE for small protein molecules (MW= ca. 6~70 kDa).
To prevent the small molecules dissolved in liquid, I performed fixation process by 5% glutaraldehyde (for 30 min), followed by 3 times water-wash (30min each). And then stained the gel with Coomassie R (45% MeOH+10% glacial acetic acid); destained with 10% MeOH+20% glacial.
Here's my problems :
1. Why the Coomassie is so hard to destain? Is it the fixation process or other reasons? (the gel in the picture had been destained for over 18hrs!)
2. If Tris-tricine SDS-PAGE, instead of Tris/Glycine, would get a better result regarding the destain difficulty? (Sorry for asking this, but I don't have materials for Tris-tricine at hand now.)
3. Why there still are some ladders (lowest MW 2 bands) missing, even though fixation had been performed? Simply because low contrast, or other reasons? (there should be 8 bands total)
I am looking forward to your kindly feedback. Thank you.
Hi Caleb :)
So, if you search for some SDS-PAGE gel results, you will see that all of them look quite blue after destaining. I guess if you expect the gel to become totally clear that would need special treatments and reagents. But I can share my own experience with you. After I run the gel, I keep it in the gel staining solution (same as yours) and put it in the microwave for 40-60 seconds (keep a lid on your container and be prepared for a strong acetic acid smell all over the lab), let it cool under the laminar and stay for 20 more minutes, pour out the staining solution (you can re-use this a couple of times), wash thoroughly with dH2O (yes only water!), pour out the water, put more water and keep in microwave again for another 40-60 seconds, rest for 10-15, and a final wash before taking the picture.
If you have enough time to leave it over night for destaining you don't have to microwave it. and you can keep washing with water till you get desirable colors. for me this method works perfectly fine and it's cheap and easy too.
regarding to the ladder issue i need more info about the sizes of those bands and which ones are missing.
Hope this could help
Cheers
-Shirin
Calib,
Tris-Tricine with Urea works quite well for small proteins - up to 10kD or slightly less. I used to use either 16.5% AA or 10-20% gradient gels.
Coomassie Blue does destain but you will need to raise CH3-OH to 25-40%, 10% will not do a good enough job. Do not overdo it for too long or you may destain too much.
Thank you for your generous sharing, Dr. Mirlohi. I would definitely try your protocol.
For the ladder issue, it was the last two (5 & 3.4kDa) that hard to identify. (The ladder is from Thermofisher #26632). I also tried the glutaraldehyde fixation process recommended by product tech support, but didn't get satisfactory result (even harder to see the bands).
https://goo.gl/8wHDYM
Thank you for your kindly feedback regarding the crosslinking facts, Dr. Alam.
I will modify the destain solution into higher percetage of MeOH, which seems to be rational way to destain more effectively.
hi,
This is what Iam using and seems to work well for me
Fixing- 50% methanol and 10% glacial acetic acid- leave it for 2- 3 hrs
Staining- 0.1- 0.4mg in the above solution. preferably overnight
Destaining- 40% methanol and 10% glacial acetic acid
Let me know if it works
Good luck,
Jaya
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