37kb and 7kb protein in western blot - (May/14/2015 )

tca should be good for removing all of the buffer components. you can confirm this by adding tca to some buffer and determining if it gets turbid or if you can pellet a precipitate.

you're blocking with milk, are you using a biotinylated detection system? if so then you'll want to replace the milk, it contains biotin.

the proteins should fix to the membrane without having to boil.

One problem with tca precipitation is that i usually dissolve directly in tris tricine sample buffer directly. And store at -20c. When i am ready doing gel run, i take it out thaw and add equal volume of sds plus bME. Generally 5 microliter of protein sample and 5 microliter of sample buffer with Beta ME. And then load it. Since I see a huge pellet in the tube after precipitation i m hoping that I have enough protein in there. I generally start with 200 microliter of extracted orotein and dissolve in 40 microliter. The conc is 3.5 microgram per microliter so literally i am dissolving 700 microgram in 40 microliter. I maybe losing some while i am precipitating. I do this because i am not aware of any better sample solubilization buffer to do the Bradford again after tca precipitation. I found this solubilization buffer 8 m urea, 4 % chaps, 1% DTT but i afraid to use this. The tris trcine sds page protocol by schagger mentions of 3 times diluted sample buffer for dissolving but it has 12 % sds and even if we dilute it by 3 times , sds would still be more. The sample buffer for triis tricine from biorad has 2 % sds only. But as I said, after dissolving in sample buffer i cannot do Bradford.
So just wondering, am i messing up any step here? Or what could be a best solubilizing agent for low mw protein such that I can do Bradford after tca ppt also to know how much protein is going in?

i used to completely dissolve the tca pellet with sample buffer with sds and 2me. you may be able to more completely dissolve the pellet by homogenizing with a pestle or sonication (prior to addition of sds) then incubating at 60-70C for 10-20 minutes after addition of sds and 2me.

there are protein assays available that work with detergent and reducing agent present (sort of). look at this webpage from pierce. just don't add the tracking dye until after the assay.

When i did Bradford then i came to know that my tca precipitated protein has far less concentration of protein than i expect ed. Partly because all of the pellet is not dissolved in there. We do not have sonic action machine. I have to rely on solubilizing solution and it does not dissolve completely. I am too afraid to heat above 60 but its so hard to dissolve even at 60.

don't be afraid to heat above 60C in the presence of sds and 2me.

you can try a pestle like this one from fisher to disperse the pellet prior to incubation with sample buffer.