Problems with protein estimation and western blots - (Feb/14/2012 )

hello everyone,

I have been suffering from western problem since last 5 months. I am using SV40 transfected cell line and looking for integrity of a complex. I started with 50 ug of protein to look over the bands intensity of the components of my complex, it seems to be ok, and then I started checking internal positive control as TBP, I found irregular bands and then I thought there may be dysregulation of TBP, as the cell lines I am using have mutation in TFIIH ( transcription factor). so i have now started to look over B- tubulin, but unfortunatlay could not see a any band for tubulin, as if I havn't used up secondary Ab...

I am also discribing here how I measure my protein samples__
I made a stock of BSA, prepared serial dilutions of 0.1 ug/ul - 0.8 ug/ul (100 ul each) and used 10 ul of these tubes in cuvettes added 1 ml of 1:5 diluted Bradford reagent (BR). I prepared 1/10 dilution of my sample, took 10 ul of it and added same 1 ml of BR, while mentioning in the spectrophotometer, i entered conc unit in the form of 0.1 mg/ml- 0.8 mg/ml,
so i m getting all my sample concentration in the range 0.2-0,5 mg/ml,,,,,,and then multiplied them by my dilution factors for each samples.
here I want to be clear that the dilution after adding BR makes any difference . As i think, it is same in all, 1 ml...if I am doing here anything wrong , please do advise me ....

Please advise me what I can do more to improve my experiment...

Thanks,
Samit

If you are using Biorad's PROTEAN 3 mini then 10-60ug total protein in each well is enough.
This is my protocol to determine protein conc.

Bradford assay
The concentration of protein was quantified by Bradford procedure (Bradford, 1976) with little modification to be able to perform in round-bottom Nunc-immuno 96-well plates (Nunc, #475434). The Bradford reagent was prepared as follows: 10 mg of coomassie brilliant blue G-250 was dissolved in 5 mL ethanol and then 10 mL of 85% phosphoric acid was added. The solution was topped up with water to 100 mL, shaken and passed through 0.45 mm filter. This was repeated if necessary until the solution turned light brownish. The reagent was stored in dark place at 4°C until use. Meanwhile, 1 mg of bovine serum albumin (BSA) was dissolved in 1 mL of PBS (1 mg/mL) and kept in -20°C until use. All measurements were run in triplicate. First, 19 mL of PBS was added into the wells followed by 1 mL of protein sample and gently pipetted up and down to mix. Immediately a serial dilution of stock BSA and PBS as standard was prepared in 6 wells (well 1: 20 mL PBS, well 2: 18 mL PBS + 2 mL BSA, well 3: 16 mL PBS + 4 mL BSA, well 4: 14 mL PBS + 6 mL BSA, well 5: 12 mL PBS + 8 mL BSA, well 6: 10 mL PBS + 10 mL BSA). When done, 200 mL Bradford reagent was added to all wells by reverse-pipetting method to avoid formation of bubbles. The mixture was incubated for 5 min before reading under microplate reader machine at 595 nm. If the readings were above 1.0 then the samples were diluted 10 times in the same lysis buffer and run again. All readings were added into Microsoft excel and further analyzed. BSA standard curve was drawn and all protein concentrations were calculated according to the trendline.