I am trying to cross-link two subunits of a sodium channel in oocytes.
Can I crosslink in intact oocytes with M2M? Is it okay to crosslink in membrane fraction? When I immunopercipitate one subunit that was crosslinked, I get a smear. It is also challenging to solubilize the membrane pellet.
Yolk fat and proteins are such a a hassle
Any thoughts, please?
thank you
have you thought of using nanodiscs?
i think this paper will be usefull "Rapid transfer of overexpressed integral
membrane protein from the host membrane
into soluble lipid nanodiscs without previous
purification"
nanodiscs would be nice, I don't have experience using them however. I maybe able to try that in the future. For now, I need a feasible method (without buying new reagents).
thank you Jihad
what are you currently using to solubilize your membrane? There are some protocols used in solid state NMR that extract membrane proteins from E. Coli extract but are able to keep it in the native environment.
The supporting info of the article bellow has one protocol, but I think if you look into the solid state NMR literature you will find many others.
Angew Chem Int Ed Engl. 2012 Aug 13;51(33):8383-6. doi: 10.1002/anie.201204666. Epub 2012 Jul 13.
M2 proton channel structural validation from full-length protein samples in synthetic bilayers and E. coli membranes.
Miao Y1, Qin H, Fu R, Sharma M, Can TV, Hung I, Luca S, Gor'kov PL, Brey WW, Cross TA.
I assume by your mentioning yolk proteins that you are working with Xenopus oocytes, correct? If so, I recommend you read Volume 36 of Methods in Cell Biology, 1991. It is entitled "Xenopus laevis: Practical Uses in Cell and Molecular Biology." If your library doesn't have it you can get it by interlibrary loan. Volume 36 has several excellent chapters on working with Xenopus oocytes. See especially Chapter 7, Biochemical Fractionation of Oocytes, by Janice Evans and Brian Kay. It provides excellent information, including a clever and very effective way to get rid of yolk proteins by freon extraction. Also in it they cite a method for isolating of plasma membranes from Xenopus embryos (Bretzel G et al. (1986) Isolation of plasma membranes from Xenopus embryos. Roux's Arch Dev Biol 195, 117-122). The Bretzel paper is for embryos, and the fertilization envelope surrounding them will be tougher than the coelomic envelope of oocytes or the vitelline envelope of ovulated eggs (see Chapter 12 by Hedrick and Hardy), but there's a good chance the method will work also for oocytes and eggs.
Also, to your specific questions about crosslinking, M2M is unlikely to work in intact oocytes. M2M reacts with free sulfhydryls of cysteine, and any extracellular cysteines in your proteins are very likely to be in disulfide bonds. Whether M2M will work in isolated membranes will also depend on presence of intracellular free sulfhydryls that are not only accessible but also in close enough proximity to each other to be crosslinked. You would likely be better off using an amino-reactive crosslinker. The smear you're seeing may indicate that you're using too much crosslinker, so are crosslinking everything under the sun. First pick a crosslinker with chemistry appropriate for your specific target protein; if you want to work with intact oocytes (a good choice) examine the sequences of the extracellular loops of your target proteins to see what functional groups are present (there will almost certainly be amino groups of lysine and arginine). Then test a range of crosslinker concentrations to optimize.
Thank you Joana and Daniel for the very helpful tips.
Daniel, I did indeed look at the freon extraction protocol. I will give it a try when I have access to freon.
So, for M2M treated intact oocytes, how can minimize reduction of the crosslink during lysis? I was thinking of an oxidizing agent like Cu:Phe?
If you are concerned about reduction of the crosslink by naturally occurring, intracellular reductants like NAD(P)H released upon cell lysis, I think your idea of using Cu:Phe should work. You could also use oxidized glutathione (GSSG). But I'm still not clear on why you've chosen to use a sulfhydryl-reactive reagent to crosslink extracellular portions of proteins.
Sorry Daniel, I didn't explain that I made cysteine mutants of two subunits in the extracellular region to crosslink them with M2M, does that make sense?
Also do you think crosslinking membrane fractions at 4 degrees celcius or on ice is better for 15-30 min?
thanks a lot
I wondered if you were working with engineered proteins, so yes, the M2M makes sense in your application. The M2M shouldn't be very reactive toward other extracellular proteins, which should give you both more leeway in the crosslinker concentration and also better signal to noise.
There should be little to no difference between doing the reaction at 0 C (ice) and 4 C. Have you considered room temp? Xenopus eggs are routinely fertilized at 20-25 C, so membrane fluidity is obviously 'normal' in that temperature range. Your proteins' proximity and diffusion in the plane of the lipid bilayer, and therefore amenability to crosslinking, could be dependent on fluidity.
thanks Daniel!! I am running the crosslinking experiment today, hopefully it works.
I am worried that at room temperature proteins will degrade?
I would not be too concerned about degradation in your experiment for several reasons. First, in intact cells protease action is controlled by sequestration, inhibition, and preservation in zymogen form. Also, any active proteases present would essentially be competitively inhibited by the high concentration of other oocyte proteins present. Indeed, if you think about it, no intact cell can afford to have active proteases floating around and degrading its cellular proteins to any appreciable extent. Protolysis and other degradative processes become a greater concern when you lyse cells, which results in mixing of enzymes previously sequestered in organelles (e.g. lysosomes) with potential substrates from other parts of the cell.
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