2 replies to this topic

[DeeDee]

Hi,

I'm trying to purify my protein from bacterial membranes through an IP. I use PBS with Bme amd PMSF as the buffer for cell disruption and then do ultracentrifugation for purifying the membrane fraction.My problem is that after obtaining the membrane pellet, it gets rather tedious to make it form a uniform suspension in the above mentioned buffer through pipetting or vortexing.Can someone tell me a better way of resuspending/solubilising bacterial membrane fractions wrt to alterations in the buffer conditions or by using any other mechanical(?) means of membrane solubilisation..

Thanks.

[protolder]

DeeDee, on 29 March 2011 - 09:03 AM, said:

Hi,

I'm trying to purify my protein from bacterial membranes through an IP. I use PBS with Bme amd PMSF as the buffer for cell disruption and then do ultracentrifugation for purifying the membrane fraction.My problem is that after obtaining the membrane pellet, it gets rather tedious to make it form a uniform suspension in the above mentioned buffer through pipetting or vortexing.Can someone tell me a better way of resuspending/solubilising bacterial membrane fractions wrt to alterations in the buffer conditions or by using any other mechanical(?) means of membrane solubilisation..

Thanks.

Once having the membranes in the pellet and eliminated and washed soluble proteins you have to solubilize membranes with any detergent as triton x100 or any other specific for that.Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins
Gopal J. Babua, Debra Wheelera, Oscar Alzateb and Muthu Periasamy, . I have seen this refence that work solubilizing integral memb. proteins more difficult to extract. urea helpstoo. Buena suerte

[mdfenko]

and you can use a dounce homogenizer.