I have purchased native gel and I am planning to use rat pulmonary artery smooth muscle cell. I would like to know what type of lysis buffer should I use to lyse the cells and prepare the final samples to load on the gel. I am mainly looking to measure oxidized and reduced forms pf thioredoxin protein. I have come across some papers from dean Jones' lab but I wanted more information from anyone else who has hands on experience regarding this. All inputs will be greatly appreciated. Thanks.
You may try "lysis buffer free" method. Resuspend your cells in PBS with protease inhibotors, disintegrate by several pulses of ultrasound (cool on ice), spin down debris and load the supernatant.
Or try "TE 50/2" (ice cold) by sonication
50 mM Tris-HCl pH7.5; 2mM EDTA + protease inhibitor coctail
thioredoxin is so small that you don't need high salt to extract it into the aqueous phase, and salt can influence the running of a native gel. I think you will get nicer bands without salt.
In my view you could use any lysis buffer or sonication treatment. Actually I always preferred the sonication in my studies. Just avoid of the using SDS or LDS in any of buffers for preparation native PAGE samples.
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