Protein extraction for SDS-PAGE from human monocytes

Wednesday, May 18, 2005

Description
Protocol for rapid extraction of undegraded proteins from cultured human monocytes for the purpose of immunoblotting

Procedure
Isolate PBMCs from Buffy Coats as usual, and select CD14+ monocytes using anti-CD14 magnetic beads (see protocols from Miltenyi Biotec).
Culture monocytes overnight in RPMI plus 10% FCS plus 25ng/ml human MCSF at a density of 4 million cells per well of a six-well culture dish.
The following day, after any treatments on the monocytes, add 5ul PefaBlock (from Roche) per well 5 minutes prior to harvesting, chill the plates on ice, aspirate off medium and add 150ul regular SDS-PAGE sample buffer (including protease inhibitor cocktail (from Roche). It is important to add the sample buffer immediately after aspirating the medium - work one well at a time. This avoids the possibility of rapid artefactual phosphorylation/dephosphorylation events prior to protein extraction. Scrape the cells thoroughly for about 10-15 seconds, recover the extract to an eppendorf tube, vortex for a few seconds, spin at 9000 rpm for 2 minutes, recover supernatant to anew tube, and boil this tube for 5 minutes. Load 15ul per lane of a Criterion (Biorad) imm thich gel. This should be in the region of 30-50ug protein.

Recipes

Supplies

Tips
PefaBlock is a cell-permeable protease inhibitor, a good alternative to PMSF, since it permeates the cell prior to harvesting.

(责任编辑：泉水)

搜索

热门标签:

Protein extraction for SDS-PAGE from human monocytes