I have resuspended the pellet in 100ul of lysis buffer. From 100ul i took 60ul for Tca/acetone precipitation.After two washes with acetone my pellet was very loose so while removing supernatant pellet also cme in supernatant.
Hence again i did centrifugation at 12000rpm for 5 mins at 4 degree.My pellet was very very small like bits. Then i estimated protein estimation by BCA.It was almost neglible.I got 0.02ug/ul.
Should i have taken whole of 100ul pellet???
I did this precipitation to remove the interferring substances- It has 200mg/ml of DTT,250mM sucrose and 10mM triethanolamine.
Sample preparation depends on the origin of the cells or the tissue. The first step is usually homogenisation or sonication followed by protein precipitation and solubilisation in a suitable buffer. Chloroform, methanol, acetone and TCA are commonly used as protein precipitating reagents. When used chloroform/methanol method larger concentration of hydrophobic proteins in specific cells pellet determine a very low proteins recovery, this because hydrophobic proteins are relatively difficult to dissolve in aqueous solutions. Precipitation using acetone produced better results than those obtained using the chloroform/methanol mixture. Precipitation with either TCA/acetone or TCA lead to approximately twofold lower recovery compared with acetone precipitation. Especially, in case of TCA precipitation, protein loss is probably due to incomplete solubilisation of the pellets and the acetone wash step. A portion of the pellet remain insoluble despite the use of additional re-hydration buffer. It is important to emphasise that the recovery of proteins is strongly dependent on pellet solubilisation. The solubilisation of protein pellets after precipitation with methanol/chloroform take 30-60 min, and with acetone and TCA, it take 90-120 min. With acetone/TCA, solubilisation required approximately 180 min. Is very quick and with increase protein recovery, when sample, in closed tube placed in ice bath, sonicate it after TCA precipitation. Better solubility may be achieved by intense and longer mixing or vortexing; however, it is crucial to avoid excessive foaming and temperature increases (ice-bath), which may cause protein degradation. Using human biological fluid as plasma or urine (see you : [Only registered users see links. ]), TCA/acetone or acetone alone precipitation, as well as ultrafiltration (Zagami F., et. al.: Proposal new technique of urinary transferrin concentration for follow-up urinary tract infections. Clin. Europ. n° 3, Year XXV, May-June 1986), yielded higher protein recoveries, but TCA was not efficient for precipitation of high molecular-weight proteins. However, as above mentioned, the precipitation methods strongly depend on the starting material.