Hello,I've been trying to use this protocol to sub-fractionate...

protocol Protocol: Isolate and Sub-fractionate Cell Membranes from Caulobacter crescentus
Hello,

I've been trying to use this protocol to sub-fractionate membranes from Caulobacter crescentus, but have not had good luck so far. I'm not getting very clean separation. The inner membrane fraction is found over a wide range of fractions (almost the whole gradient). The o
Read more Read less more less
answer Answer
recommend recommend recommend recommend Recommend
follow follow follow follow Follow
share share Share
2 answers
Sort by: most helpful / most recent

June Javens-Wolfe Answered Aug 20, 2013

Indiana University

Thanks! I'll try that. I had set the deceleration pretty low, but I didn't adjust the acceleration speed.
0 helpful
0 unhelpful
comment 0 comments down up
share Share

Khatira Anwari Author Answered Aug 19, 2013

Monash University

Hi June,

After reading about your problem, the first thought was that the sucrose gradient has been disturbed. This can happen mainly when it is being prepared as you stack sucrose solutions to make the gradient or when the sucrose gradients are centrifuged. After you pour the gradients, you should be able to delineate the different concentrations of sucrose by holding the tube to light to see the translucent boundaries. Also, before you spin the sucrose gradients at 34,000 rpm in the ultracentrifuge, make sure you set the acceleration/deceleration to a slow speed. On the Beckman ultracentrifuge that I was using, I would set the acceleration/deceleration speed to 5 (with 9 being the maximum). This ensures that the sucrose gradients are not disrupted by sudden high speeds. Considering your inner membranes and outer membranes are near the bottom of the tube, it is likely the run is started or stopped too quickly.

I do not think the lysis conditions are too blame, as long as you are using somewhat consistent pressure to bust the spheroblasts.

Cheers,
Khatira


0 helpful
0 unhelpful
comment 0 comments down up
share Share

My answer

Write your answer...

References (optional)

add Add more

post Post an Answer