I want to check the mTORC activity and for that I am using rat kidney. I used cell lytic buffer from Sigma Aldrich to extract the proteins from rat kidney (50mg sample homogenized in 200ul cell lytic buffer with Halt protease and phosphatase inhibitor cocktail followed by centrifugation at 13000 g for 15 minutes), took the protein concentration by BCA assay, and then ran SDS-PAGE at 80V for 1.5 hours. I used BioRad's 10X Tris/Glycine/SDS electrophoresis buffer. I then did the transfer to PVDF at 0.18A for 1.5 hours. I used 10X Tris/Glycine as a transfer buffer. I incubated the blot with S6 primary antibody at 4 degrees overnight, washed with TBST 3 times and incubated with secondary antibody for 2 hours at 4 degrees followed by TBST wash 3 times.
When I developed the membrane, I see the bands are not aligned. I see it smeared. Can anyone help me point out what could be the issue? Shall I lower the voltage while running the gel? or the problem could be something else?
That indicates an uneven distribution of the current when running the gel. Check the condition of the electrodes in the gel tank. Alternative causes can be air bubbles when preparing the gel, incorrect volumes of running buffer, cracks in the tank causing buffer to leak and earth.
Dear Sachin Aryal
i think that you can try to run an other gel by just diluting the samples in cell litys 4 or 5 timers with a buffer that do not contain any detergent (eg TBS or PBS) and then load it in the SDSpage, since i suppose that the bad behavior of your gel may be due to the combination of 2 different aspects
1) Sample overloading
2) Presence of a detergent different from SDS (i think is chaps) in the Cellytic
by diluting the samples you can load less amount and reduce the detergent % at the same time.
good luck
Manuele
Definitely looks overloaded. Find the right concentration that provides a clean lane. Choose one sample, and dilute 1:1 in a series starting at the concentration you show above and performing 6 - 8 dilutions. Ideally, you will find the dilution that gives you a non-saturated cleanly run lane. Then go back and use that same dilution/concentration to repeat all of the samples.
In general, it's best to run ranging pilot experiments to determine your dynamic range. You have to be able to quantify/interpret an effect and you are not able to do so with saturated blots. Give it a try and best of luck.