immunoprecipitation of membrane protein from gram+ bacteria - (Dec/22/2005 )
Hi every body
New in the field of microbiology, i am wandering if it is possible to tag bacterial proteins with myc, ha and so on and do co-ip. My project is to to understand the functional organization of an ABC transporter expressed in gram+ bacteria. Does anybody have any clue + how to extract membrane proteins from gram+ ???
Thanks in advance.
-delphinelechardeur-
To isolate membrane protein from Gram positives you may want to look at this article.
http://www.pubmedcentral.gov/articlerender...ubmedid=3015800
QUOTE
The cells were washed twice with cold water and suspended in osmotic buffer (0.4 M sucrose plus 0.075 M Tris [pH 7.5]and 2.0 mM MgSO4). The cells from 1 liter of culture were suspended in 40 ml of buffer, which was then transferred to 160 ml of buffer previously warmed to 37°C and containing 0.5 g of lysozyme and 2,000 U of mutanolysin (Sigma Chemical Co., St. Louis, Mo.). The suspension was then incubated for 2 h at 37°C with gentle shaking.
The resulting suspension was centrifuged at 9,000 x g for 20 min at 4°C. The pelleted cells were suspended in membrane buffer (50 mnM Tris [pH 7.5] plus 10 mM MgSO4), and NaCl was added to give a final concentration of 0.8 M. Shortly after the addition of the NaCl, the cells lysed, and
the suspension became viscous. DNase and RNase (10 ,ug of each per ml) were added, and the suspension was incubated for 45 min at room temperature with gentle swirling. The inhibitors 6-aminohexanoate (40 mM) and p-aminobenzamide (6 mM) were added initially to reduce protease
activity.
Membranes were pelleted by centrifugation at 35,000 x g for 30 min at 4°C and were washed twice with 30-ml portions of membrane buffer. They were stored in pellet form at -20°C. Before use, the pellets were thawed and suspended in membrane buffer
The resulting suspension was centrifuged at 9,000 x g for 20 min at 4°C. The pelleted cells were suspended in membrane buffer (50 mnM Tris [pH 7.5] plus 10 mM MgSO4), and NaCl was added to give a final concentration of 0.8 M. Shortly after the addition of the NaCl, the cells lysed, and
the suspension became viscous. DNase and RNase (10 ,ug of each per ml) were added, and the suspension was incubated for 45 min at room temperature with gentle swirling. The inhibitors 6-aminohexanoate (40 mM) and p-aminobenzamide (6 mM) were added initially to reduce protease
activity.
Membranes were pelleted by centrifugation at 35,000 x g for 30 min at 4°C and were washed twice with 30-ml portions of membrane buffer. They were stored in pellet form at -20°C. Before use, the pellets were thawed and suspended in membrane buffer
To isolate the membrane in gram-positive you need to remove the cell wall using enzymes. Lysozymes is the usual one but certain gram-positive are less sensitive to lysozymes so you need to add another enzyme that will digest the cell wall.
You can also add glycine during the growth of you bacteria. Glycine get inserted in the cell wall and it becomes more sensitive to enzymatic digestion.
-iansmith-
Hi Ian,
Many thanks.... for the help 
Merry christmas.
-delphinelechardeur-