For radioligand binding assays, our lab, for years, has used the following protocol for preparation of membrane.
1) Centrifuge cells (HEK 293 and CHO-K1) at 12,000 g and 4 C for 10 min following transfection of cDNA encoding GPCRs
2) Decant supernatant and resuspend/lyse cells in ~25 mL 50 mM tris-HCl (pH = 7.4) by rigorous vortexing or shear force using a syringe
3) Centrifuge cell membrane at 12,000x g and 4 C for 10 min
4) Repeat steps 2 and 3 one or two more times depending on the size of the membrane pellet.
5) Store pellet at -80 C
We have noticed that the cell membrane aggregates sometimes during the 2nd or 3rd round of vortexing, and if not then, sometimes upon homogenization after freezing. Because of this, the preparation cannot be used in a binding assay, and after filtering out the clumps of membrane, there is no specific binding.
This seems to be specific to HEK cells (transient and stable transections) in some plates and has never occurred in CHO membrane. Even upon obtaining new HEK cells from another lab, the same thing occurs and there appears to be no sign of contamination in the cells or media (media is not quick to be spent, no turbidity, no visual indication of contamination under a microscope, cells appear healthy). This phenomena also appears to occur at random, only in some plates, even when the tris-buffer is sterile filtered.
Dear Dr. Singh, thank you for your help. We used the benzonase endonuclease and it seems to have fixed the problem. However, this only works when the membrane prep is incubated at 37 C for ~20 minutes between steps 2 and 3, which concerns me regarding the stability of the GPCRs and proteases in the preparation. Hopefully we can get it optimized to work at cooler temperatures with shorter incubation times. Nevertheless, your help has given us a good place to start!
What really confuses me is that CHO cells do not give this problem, and I cannot remember a time before a year ago where membrane clumping was an issue (even in HEK cells), at least not nearly has bad as it has become recently. Another confusing thing is that it only happens to some HEK membrane preps, so I am having a hard time narrowing down the cause of the DNA/membrane aggregation.
As it turns out, I was able to circumvent this problem with another method of membrane homogenization. By using a tissue grinder rather than vortexing the cells, no clumps occur. I suppose the tissue grinder does not break the nucleus and the vortexing does, so no DNA is released to aggregate with the protein.
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