Hello, sometimes when it doesn't run well could be a leakage of the current (or some electrical problem, check if everything is right ), another option could be bubbles under the mirror. but if this issue is happening frequently you should try to reduce the gel concentration a little bit, 11% or 10% I.E. other solution could be to run it for more time(2 hours) at low temperature to avoid heating

Hi Zamani,

I think you need to check your running buffer preparation because if your running buffer is too old(prepared for a long time) could result to that.1. Prepare a fresh running buffer and loading buffer. 2. Carefully check your running buffer protocol and composition.3. I will suggest that you run your SDS-PAGE with 100V for a good resolution.4.During course of your gel setting,carefully observe your pipetting of your recipes such as acrylamide( pipette error could affect your acrylamide composition of the gel).

Finally,if i may ask what amount of your marker/proteins did you load in each wells?

I hope this hints will help you solve your problem.

All the best.

Thanks for the reply Angel and Adesola.

The buffer was made freshly. However, the acrylamide took around half and hour to get solidified completely. Its was a bit longer than expected.

I have added 8uL of temed and 50 uL of 10% APS for 10 mL of 12 % separating gel. For stacking gel 4uL Temed and 35uL of 10% APS. was used.

I loaded 7 uL the marker and 10uL of different concentrations of my samples.

Thanks again. I shall consider your recommendations.

mah

I think the problem could be due to non-uniform cross-linking of separating gel. Follow the tips given below.

1) Use 1% APS for separating gel

2)Degas the acrylamide :bisacrylamide solution for 2 min and incubate in ice for 30 min before adding TEMED and APS for crosslinking.

The cross-linking will take around 1hr for 7cm gel but you will get uniform gel.

Hope this might help.

Thanks for your suggestion. I may consider that too.

I dont see a stacking gel and the gel wells: were these removed? It might help to diagnose the problem.

My initial thought was upper tank buffer leakage, which is difficult to see with some tanks, but you say that isnt the problem. Another is that the power supply failed after a certain time.

That very long polymerisation time is possibly down to APS: I make fresh 10%, aliquotise and freeze at minus 20: they keep for years!

I always use prestained markers: these reveal any problems during the gel run and can save a lot of time.

Note that I stopped degassing PAGE about 2 decades ago.

Hello if your problem isn't resolved, try to change your running buffer and/or your gel buffer. SDS concentration could be too low.

Hope it will help you.

Hi,

If your gel takes a longer time to polymerize (solidify) that means your 10% APS is very weak so you need to prepare new 10% APS solution.For example if you use Bio-Rad for 12% SDS-PAGE.....resolving gel is always prepare 4ml (use 20ul of APS for resolving and 5.6ul of TEMED) and 2ml of stacking gel(use 10ul of APS for resolving and 2.8ul of TEMED).Within 15-30mins it will polymerise for both.Based on the volume,its ok because 10ul is small volume .I think from the beginning of the experiment that you observed the gel took a long time to polymerize,you should have take note that there might be error at the end.Try new experiment with good condition and lets see the outcome.

Regards

Thanks everyone. Dear Geoff, APS was prepared few months back and distributed in 1.5mL tubes, stored at minus 20. However, I might have used the same tubes for several times and, melting issue.

I have made a new one and going to use it.

The marker i used was pre-stained marker. so, the last two lanes are not separating. as i mentioned before, the voltage was 100V but the current was so low, about 0.09 mA.

I though it might be the running buffer. so made everything fresh and will see what happens next time. The protocol i used for a 10X running buffer was: 15.1 gr Tris base, 72gr of glycine and 5gr of SDS dissolved up to 500mL.

I might have used less glycine and more SDS. Now prepared 10% SDS and used 50mL in 500mL of buffer, 94 gr of glycine and same amount of Tris.

Might this cause the problem?

plus that I see my desired protein bands which should be at 25-26kD are close to 20kD! Can it be for the same reason? or my protein might have been cleaved or degraded? I should mention that the positive control (the very first well) is like my sample at the lower position too,20kD.

I expressed the protein in E.coli. So after inducing overnight. I boiled the bugs for 8 minutes and then pelleted and added 5X SDS loading dye buffer.

Thanks a lot.

Dear Geoff, yeah the stacking gel was removed after running being done. The samples were quite well at the bottom of the wells and run well through stacking gel.

I dont understand why, if you are running prestained markers, you chose to stop the electrophoresis when you did. OK, so its taking longer than normal, but that doesnt matter, what does is the separation, and that was under your control.

The issue still is there even after using fresh buffers. I have let it to reach the edge of the gel but still the last two lanes of the marker have not been separated or are very close to each other.

I did western bloting and it confirmed my expression very nicly. However, I am not sure if my protein have been cleaved or degraded which stands lower than 20kDa while it should be 25 to 26kDa? or it is simply the marker problem?

I know i probably have to check another marker but was thinking about chechinkg my protein too.

Any suggestion on the methods for checking the cleavage of the protein?

Thanks all,

We had similar problem with electrophoresis and it turned out it was a water quality problem. Check the conductivity of the water you are using. Our system for making deionised water started to malfunction and after replacement everything was back to normal.