We are running subcellular fractionation of transfected cells with our protein in question to determine its localization. The protein is HA tagged and we are running the fractions on a western blot and blot with anti HA.
The cytosolic fraction results in a very clear band in the expected size. However the membrane fraction blot results in a smear through the length of the run. They're on the same gel so it's not a problem with the gel or antibody, my guess it's related to the sample itself. something specific to the membrane preparation.
Does anyone have an idea what happened? was the sample degraded? is there a specific problem with the membrane fraction? any suggestions what to do? Of course we used PI and DTT.
Thanks!
Hi Pazit,
Sometimes, membrane proteins are highly glycosylated that's why it might appear to be smear on a western blot gel.
Also, you might want to try not to boil or heat the samples before running the gel. Just add sample buffer. If the samples are too viscous, you can add DNase or Benzonase, or like Eda said to mild sonicate it. Since you are using fractionated sample, I doubt the sample will be viscous. Good luck!
How are you running the membrane fraction?
Are you using first detergent extraction. What amount of protein you are loading?
All these factor may affect your Western results
Hi Pazit,
What is the approximate protein concentration of the membrane fraction? How much are you loading on the gel? Too much protein (membrane-associated proteins, probably) causes smear. Also, is your membrane fraction more viscous than the cytosolic fraction? Maybe you can try mild sonication before boiling.
Hi Pazit,
Sometimes, membrane proteins are highly glycosylated that's why it might appear to be smear on a western blot gel.
Also, you might want to try not to boil or heat the samples before running the gel. Just add sample buffer. If the samples are too viscous, you can add DNase or Benzonase, or like Eda said to mild sonicate it. Since you are using fractionated sample, I doubt the sample will be viscous. Good luck!
Hi, Please dilute your membrane fraction , centrifuge and then check protein load the gel. I hope this will solve your smearing problem.
Good luck
Hi~~
I have met the similar problem with you when I run the blot of Glucose transporter protein. This may due to the protein PTM such as glycosylation or the denaturing condition. Finally, I have solved the question with the experience from Abcam antibodies data sheet. you may have a try to denaturing the sample at 70 degree but not 95 for 10 minitunes or prepare the samples with TCA immunopicitation, I hope this ma help you for your progress. Good luck!
Please try different membranes and apply a range of concentrations of your extract. Remember to block your membranes before use.
- Badges
- Science method