Hello Chiara,

Most likely there is something wrong with your electrophoresis buffers. I would suggest to make them fresh, as described in the original publication of U. Laemmli, Nature 227, 680-685. While doing so, make sure that your pH meter is properly calibrated and that the SDS stock solution contains no crystals, as this indicates that it has been hydrolyzed and contains sulfuric acid.

Dear Pierre,

Thank you very much for your reply.

I will double check the pH of the running buffer (I made all solutions last week) and the SDS.

Kind regards,

Chiara

HI Chiara, I don't think your lysates are the problem. Something tells me that either your Acrylamide is off, or its concentration is off. I looked up the MW markers you mention to see what they should look like, but I couldn't recognize what those bands are present in your gel. Regardless, the bunching up at the top is problematic. If those are lower MW markers after a 1hr run, then perhaps your acrylamide concentration in way too high.

Also, if the gel is still in the glass in the picture above, then you should have no bubbles there. Make sure the glass is very clean before pouring the gel.

In general, always maybe the APS fresh before pouring the gel, and polymerize for at least 1hr (the longer the better). Don't vortex the mixture (to avoid polymerized bubbles) before pouring, and check the pH of everything as suggested by Pierre. But I would make fresh acrylamide as well (or buy some).

Hi Chiara,

I agree with Bryen A Jordan. If my bands are diffuse or not clear (including Marker), my usual practice is to re-prepare the gel with fresh reagents.

Hope it helps!

Hi Chiara,

you can check the SDS-PAGE Machine as I experienced the same problem before and the problem was in the distribution of the electricity of the SDS-PAGE Machine,you can used another SDS-PAGE Machine and see, also you have to check your protein molecular weight and the percentage of your gel that you used ??your gel seem very thin which means very low percentage, as you know if your protein has low molecular weight you have to increase the percentage of your protein to see your bands more clearer.

good luck

Mohamed

Hello Chiara,

I agree with Mr. Hawara. Gel percentage, protein concentration, distribution of electricity, staining and disdaining time is very vital to visualize the bands. You have to be very careful in each step of your experiment.

Hello everyone,

Thank you for your advice! Despite I re-prepared all the solutions, I still have the same problem. However, I did an attempt in a different lab, with their materials and my buffers, and I have got this result (see annex). Therefore, probably Mohamed's idea could be reasonable. The protein is ZYP1 in hexaploid wheat, MW= 98 KDa and I used 7.5% SDS gel.

In addition, I tried to blot my sample with Ab raised against rat (blocking solution= TBST with 0.05% tween 20 + 5% milk. Incubation time for blocking, I and II Abs conjugated with HRP: 1h), but when I develop the film, it comes black and I only see the ladder. In this case, I tried to increase the washes and dilute the EZ-ECL, without any results.

Any clue?

Thank you very very much for your precious support!

Best,

Chiara

Hi Chiara,

its good news that you overcome the first probelm of the your gel But its still not clear which defiantly can affect the western blot results.

for westen blot , usually from my experience the main problems of Westen blots results is the ECL buffer , are you prepare by yourself or you bought it ?? If you are prepared by yourself just be sure it’s used as soon as possible to avoid the oxidation and be sure you properly distributed on your nitrocellulose paper.

if your western blot not working properly you can use different method for idnetify your protein such as Mass spec and also i have to ask did you make size exclusion chromatograph??

Primary & Secondary antibodies once change to another brand