Troubleshooting SDS-PAGE: turquoise Area after Coomassie staining - (Jan/09/2015 )
Hello everyone,
i have a problem with my SDS-PAGE. Everytime i am using a 8% Gel (as in the pictures) there appears a turquoise Area at the bottom at the Gel after Coomassie staining. The turquoise color appears while destaining with Ethanol/acetic acid and disappears while deastaining in dest. H2O. But even after the destaining you can see the Area because its a bit darker then the rest of the separating gel. The Area in wich this effect appears reaches from the bottom of the Gel till the 15kDa titled band.
But as you can see on the attached pictures it clearly interacts with the Proteins in this Area and also stopps proteins in this range passing. But im not even sure if the affected band is the 15kDa band, because in the early stage of the run, the affected band runs above the 25kDa titeld band and is only at the end of the run lower. On the other hand the 10kDa Band is inside the area but not affected (and its the 10kDa band because its green after destaining).
After i first saw this i checked my Gels while running and you can see two seperation layers (like the one between the stacking and the seperation Gel) running. The upper one runs about 1cm above the Bromphenol blue and the other one runs approx. 2mm under the Bromphenole blue tracking dye. I checked this with different Acrylamide concentrations and in doesn`t change with differend concentrations. First i used 12% gels and the area always ran out of the gel and didn`t affect the results, but now with 8% gels its pretty disturbing.
My question now is: What is this and how can i get rid of it?
The conditions of my SDS-PAGE are the following (im using the Bio Rad system):
Running buffer (always fresh prepared):
25mM Tris-Cl
200mM Glycine
0,1% SDS
the pH is 8,3 after titrating with HCl (i know its not recommendet to adjust the running buffer but thats the way i learned it and in never had problems with it till now)
2x Loading dye:
0,125M Tris-Cl
20% Glycerol
4% SDS
0,05% Bromphenol Blue
pH 6,84
(and no the Loading dye doesn`t have a reducing agent because i want the disulfide bonds in its native state)
I use for 5ml stacking Gel:
670µl Acrylamide (i use the rotiphorese Gel 40 with 29:1 Acrylamide to Bisacrylamide so the final concentration is about 4%)
1250µl 0,5M Tris-Cl pH 6,8 (so the final concentration is about 0,125M Tris)
50µl 10% SDS (so final 0,01% SDS)
5µl TEMED
50µl 10% APS (always fresh prepared)
2975µl dest H2O
And for 10mL separating gel:
4000µl Acrylamide
2600µl 1,5M Tris-Cl pH 8,8 (so the final concentration is about 0,39M Tris)
100µl 10% SDS (so final 0,01% SDS)
10µl TEMED
100µl 10% APS
3200µl dest H2O
I overlay the separating gel with 2-propanol and let it polymerise for 20-30 min. Anfter that i discard the 2-propanol with paper towels and put the stacking gel over it. (maybe a little 2-propanole is left, could it cause this?)
After making the Gels i usually leave them for 2-5 days in a fridge with wet paper towels around in a plastic bag.
For the elektrophoresis i use 150V for approx. 80 min.
The sampels contain 20mM HEPES and 10mM EDTA, but this Area also appears on lanes only with loading dye so it couldn`t be the sampels, right?
No one in my lab has never seen something like this before so maybe one of you know this phenomenom?
I would be happy about your suggestions 
Thanks!
(i also attached the used marker so its clear how it should look) 
a few things:
you're seeing a "buffer front", it is compressing your 15kda band. sometimes, as with your 12% gel, you can run it out of the gel without losing any bands but not with the gels you're running
your sds may be decomposing, try a fresh lot
do not adjust your running buffer, it worked okay for 12% and not for 8% gels (you were able to run it out of the 12% gel). make sure you use tris base (not tris-hcl)
rinse the gel with water when removing the 2-propanol, residual alcohol can cause problems
20-30 minutes to polymerize may lead to incomplete polymerization. i usually wait until there is a sharp line and squared edges before removing the alcohol.
ensure that your gel is warmed to room temperature prior to loading, sds (micro)crystals may form in the gel when refrigerated
i prefer to store without the stacking gel and stack fresh prior to use
buffer constituents of the sample can cause run artifacts, including adjacent lanes, if the samples buffer is a significant portion of the sample then you may want to consider exchanging prior to sample preparation.
ok thanks a lot!
i'll try out all of the factors you mentioned. Hope i can get rid of it.
Thanks a lot again!