From my experience working with L-VDCC I noticed that when you heat the protein sample to denature it, it forms a gunk and does not move out of the well, nor does it resolve. Try to run the sample without heating if you can.

Dear Hai,

Did you use trypsin to detach the cells? This may have denaturated the GPCR. 

Best,

Mannan

did you do protein assay before loading? to check whether you have enough amount of proteins?

From my experience working with L-VDCC I noticed that when you heat the protein sample to denature it, it forms a gunk and does not move out of the well, nor does it resolve. Try to run the sample without heating if you can.

Hi,

I think Prabodh Kumar is right, looks like the GPCRs aggregate after boiling (happens sometimes with proteins with several transmembrabe domains), so try non-boild samples.

Good luck,

Carsten

Hi Haitao,

Hope all is otherwise well. Sorry to hear about your troubles.

2 possible issues:

1. Extraction using RIPA gives rise to gooey extracts due to genomic DNA in your preps, especially if you are using high density cultures such as  293 cells. This viscous substance should not be loaded onto the gel as it will remain trapped in the well along with your proteins. This needs to be broken up and while sonication should work, I have found that passing the sample through a 30g needle repeatedly until clear, is much more effective. The solution should also contain DTT or BME. 

2. Reduce the methanol content in transfer buffer to 10 or 5% from the usual 20%. Large MW proteins >150kD have trouble exiting the gel during transfer when the methanol content is too high. You can also add 0.1% SDS in transfer buffer to help this. 

Hope this helps.

Best wishes

Hello Prabodh and Carsten

Thank you very much and we will try it again without heat denaturing. 

Haitao 

Hello Mourad,

Thank you very much for the input and the detailed protocol. 

We will follow it and give it a try.  

Best wishes,

Haitao

Hi Hai T Yuan,

For 293 cell-lysate preparation you can simply use SDS lysis buffer (1-2% SDS, 10% glycerol, 50mM Tris (pH6.8), 150mM NaCl, 10mM EDTA, 10mM DTT).

Instead of trypsinizing the cells you can add 500uL of lysis buffer per 90mm plate, collect the lysate and boil in water bath at boiling temperature.

In SDS lysis buffer most of the proteins including membrane proteins and filamentous proteins get solublized. So here heating will not encourage aggregation rather a essential step for this protocol.

This might help you.

Good Luck. 

Dear Haitao,

I face the same problem and I am curious whether you were able to resolve the matter.

I once got multiple bands using lammli buffer directly for lysis (betamercaptoE, 94°C for 15 min). I want to avoid this however since aggregates/several bands are hard to quantify and the protein would obviously not be functional that way. I tried a special membrane protein extraction kit buffer, but was not able to detect my protein with this. For GPCRs, another buffer is obviously needed. Did you try modifying the ripa buffer? I will try next week and keep you updated if you like.

Good luck!

Best wishes, Florian