I've uneven bands (size, intensity, and staining) when doing Western Blotting and I'm wondering what the problem could be.
The protocol:
-10% separating gel (0.375M Tris pH8.8; 0.1% SDS; 10% BisAcrylamide (37.5:1); 0.1% APS; TEMED) ± 15' to set
-stacking gel (0.125M Tris pH6.8; 0.1% SDS; 4.5% BisAcrylamide (37.5:1); 0.1% APS; TEMED) ± 15' to set
-proteins are denatured in Laemmli sample buffer (BioRad) +bMeOH, heated for 10' @ 95C
-samples are run for 20' @ 80V and ±1hr @ 100V
I think the problem lies somewhere here, but I'll also briefly describe the rest of the procedure
-proteins are transferred to PVDF membranes using a semi-dry blotter for 1hr @ 15V
-30' incubation in blocking buffer in TBST
-o/n incubation in primary antibody in TBST @ 4C
-3x 15' washes in TBST
-1hr incubation in secondary antibody in TBST
-3x 15' washes in TBST
-ECL
I have not experienced this problem in the previous labs I've worked, I've always had straight clear bands and plenty of experience with WB, I guess a change in reagent or something could be the reason but I can't put my finger on it. Previously I've always used Nupage LDS sample buffer and wet transfer. Other than that there's not a big difference. Any advice is much appreciated.
Hi Amila,
I think this is caused by two reasons, a) the gel is not properly prepared like homogenous or not polymerized well, b) started to run the gel with high voltage at the beginning. I strongly think, this is because of the gel, not other components.
I hope you will find the reason, Good luck..
Hi Amila,
I think this is caused by two reasons, a) the gel is not properly prepared like homogenous or not polymerized well, b) started to run the gel with high voltage at the beginning. I strongly think, this is because of the gel, not other components.
I hope you will find the reason, Good luck..
source credit: Abcam,
Gel has set too quickly while casting and the acrylamide percentage is not even throughout the gel.
Review the recipe of the gel and the addition of TEMED to the gels, add some 0.1% SDS in water to the top of the migrating gel while it sets to stop it from drying.
When you prepare the gel, after adding APS votex the mixture vigorously and pour it as soon as possible.. My experience is, mix the gel with APS in a 50ml conical tube and directly pour in to the mold without using a pipette...
Hi Amila Zuko . It could be due to particulates in your samples. Have you tried spinning them before loading?
As sometimes preparing western blot gel causes some issues like uneven bands, you can try precast gels (e.g. Novex Tris-glycine gels) that come with variety of concentrations depending on the molecular weight of the protein to be separated.
If you stain the gel without blotting it: Is the reaction also uneven? Do you wash the gel pockets before sample loading? How does the gel look if you stain it after the blotting?
When doing the Western Blotting part: Do you roll gently over the sandwich to remove the tiny air bubbles before adjusting the lid? How old is your transfer buffer and how is it stored? If you shake it, the bubbles should come up quickly otherwise there could be some growth in it which makes it more viscous.
Voltage should not be a problem, I start run with 100 V and increase to 180 V after 10-15 min once protein reaches to the separation gel and never have a problem. I would guess it is a problem with your gel - first case - gel should be polymerized properly - I give the stacking gel in the end minimum 1.5 h of time for polymerization or prepare gels and store them over night in wet paper in the fridge, not just 15 min - proper mixing of the solution is also mandatory. Washing of the pockets prior to loading with buffer is also helpful.
By the way - how about the front line? Does it look good or do you already see uneveness there? have you ever stained the membrane after blotting with Ponceau solution? this is a 1 min job to see first equal loading and second if the bands have any problem. Ponceau has the advantage that it can be easily be destained with first water and later on milk/TBST.
In my opinion, there was a problem regarding contact of electrodes, voltage source in the middle o the gel. This may be due to loosen contacts of anode/cathode plates during semidry blotting or in gel cosistency or other sources of uncertainty regarding contact to voltage sources (smudges at or defects of the electrode ...) for tank blot procedures.
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