Elution of protein form Nitrocelulose or Immobilion membranes

[scout_caro]

Hi everybody: somebody can help me ?.. .I´m triyng to elute proteins from nitrocelullose membranes, to use them in a cell culture, but the protocols that i found were to olds and i need to know if one of you know or have if there are new modifications about.

the newest reference that i found was:

Preparative elution of proteins blotted to Immobilon membranes
Analytical Biochemistry 168:48-53, 1988
B Szewczyk, D Summers

thanks for your help

[admin]

Hello there!

yes I found that reference as well:

Anal Biochem. 1985 Jul;148(1):87-92. *
Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

• Parekh BS,
• Mehta HB,
• West MD,
• Montelaro RC.

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.

[scout_caro]

thanks a lot i will try that technique....and i will say to you if it works....