Our initial plan is to use streptavidin or neutravidin coated 96-well plates (15115, Thermo Scientific) and biotinylated lectins to isolate glycoproteins from complex samples then probe for specific targets of interest. One issue is that some of our targets are part of multi-subunit complexes and we want to also make sure that we are pulling individual subunits and not the entire complex.
Our starting material is either a tissue chunk or nitrogen-pulverized tissue from postmortem human cortex. We typically dounce homogenize in either sucrose buffer (5mM Tris pH 7.4, 320mM Sucrose) or isotonic extraction buffer (ER0100, Sigma) which is basically HEPES with sucrose, EGTA, and KCl. For some downstream applications we will also either sonicate/nitrogen-cavitate during homogenization or will spike the homogenate with various detergents to break apart complexes (including isolating individual subunits of different neurotransmitter receptors).
Any advice on which buffers would work best (I'm assuming something that doesn't have sucrose) and what homogenization buffer/method to use would be greatly appreciated. Also, will detergents like SDS or triton interfere with the ELISA and are there any alternatives to break apart receptor subunits if they do?
Any SDS (and probably Triton buffers, need to be tested) will definitely interfere with your ELISA experiments. If the functional activity of your target proteins as a multimers is not important you can try to use some dissociative conditiions to get your protein dissociated for subunits.
For more information please specify the compositions of the buffers you use now, the protein of your interest, the number and nature of the subunits, and the lectin you are using for capture your target.
Dear Tony!
I studied lectins from different species of flax. I used to extract the buffer of the following composition: 20 mM potassium phosphate buffer with 0,1 M ascorbic asid, 0,35 M sucrose, 0,01 M EDTA and 1 mM Triton X-100
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