TLR4 IP
Fabiano Pinheiro da Silva
Tuesday, 04 March 2008 22:56 UTC
have you ever tried to IP TLR4 in primary cells (macrophages)?
people say it’s very difficult to get it.
I would like to try, but I’m in doubt if it worths.
Thanks,
Fabiano
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Replies
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I have tried IP of TNF-R1 and looking at the signalsome includnig the IKK complex with very poor results. I was able to pulldown TRAF2 and RIP with the receptor, but everything else was much harder. Also, to accomplish this, I was growing large quantities of MEFs, and I know macrophages are harder to work with than MEFs.
I guess it would depend on what you are trying to pulldown with TLR4. You may be able to get receptor-proximal proteins, but it would be difficult to pull anything out beyond that without lots of optimization and cells.
I would be interested to hear anyone else’s experience with pulling down receptors and looking at proteins attached to them.
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Brock,
Which’s the detergent you’re using?
I’ve IP TNFR-1 and IB TRADD last week, using digitonin 1%, but I only did that, I haven’t tried more downstream signaling. It worked well.
I haven’t found a good antibody to blot IRAK-1, so I’m planning to IP TLR4 and blot IRAK-4 and maybe IRAK-M too.Thanks,
Fabiano
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The detergent I used for lysis was 1% Triton-X 100.
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Isn’t it worthwhile to try triton 0.5% or digitonin 1% in your case? Maybe CHAPS, if you don’t want to use digitonin…
In my case, the problem is that TLR4 membrane expression is low, I’m not able to IP the receptor, I’m wondering if it worths to try, using more than 10e6 cells per sample.. -
sorry, I mean 10e7 cells per sample..
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Brock,
I’m having problems to blot phospho-JNK. It’s really hard to get a good signal. What’s the antibody that you use? At which concentration?
Are your IP working better?Thanks,
Fabiano
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Hi everybody,
phospho-JNK appears after long time exposure and high antibody concentrations.
TLR4 IP also works, but one needs a lot of cells (2×10e7 per sample).
No more questions for now.
I learned a lot during the last months with you.Many thanks, Fabiano
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