I want to cross-link the TNF receptor with His or flag tagged TNF using chemical reversible or irreversible cross linkers. After Incubating for two hours on ice with 200ng/ml TNF with 50 million HELA or U937 cells, I wash the cells and add 1 mM of cross linkers for thirty minutes on ice. After quenching the reaction with 100mM tris I wash the cells with cold PBS. Cells are lysed for one hour in lysis buffer (20mM HEPES,1% tritonX-100 ,150mM NaCl,1mM PMSF and protease inhibitors) and are spun down at 12000g to settle the debris. The supernatant is incubated with affinity beads i.e. nickel-NTA or flag beads to pull down TNF and its associated receptor as a cross-linked product. After boiling the beads in 2X SDS-sample buffer, I detect the cross linked product with western blotting using anti-tnf antibody.
I am not able to see any cross-linked product of mol wt above 70 kDa in the blot. I tried IP using antiTNF antibody or antiTNF R1 antibody also but I didn't succeed.
Hi,
I think you need the TNFR+TNF complex. Consider the following...
1. First fix the right time point. TNF binding to TNFR takes few minutes and the TNFR+TNF complex rapidly get internalized by endocytosis and in some cells the TNFR will be recycled back to cell surface or gets degraded. In either case TNF is not going to get recycled. So, at 2hrs time point you might lose TNF or both TNF+TNFR. I would recommend 5-15 minutes time point because TNF is well known to transduce it signal to the level of IkBa degradation at 15 mins (considering 200ng/ml this would be maximal. I would recommend 17ng/ml (1nM) is more than sufficient to saturate the receptors on surface (even at high density of cells).
2. Expecting a product at 70kDa range after crosslinking is not worth. TNF=17kDa; TNFR=55kDa (together 72kDa). But...crosslinking will cause linking of additional proteins from adapter complexs+receptor aggregation....the so called DISC. A single DISC might be in mega Dalton in size (I am not sure exactly how much). In some cells you'll be able to see DISC microscopically..then imagine how big the DISC will be...!!. In this case, the DISC may be lost along with the pellet fraction in your clarification step (12000g).
3. I recommend to use alternative methods to crosslinking for your type of analysis.If more info needed, plz send message in RG.
Good Luck..!!
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