Protein Extraction from cell media for western blot - (Jun/20/2007 )
I did a search for this, but I had trouble understanding the responses. Anyway, I transfected a construct containing a gene for a secreted protein into some cells. I want to extract the protein from the cell media in order to run a western blot and compare the differences in the amount secreted between wild type and transfected cells. What is the best way to do this? I searched and searched but have found little. Any help would be GREATLY appreciated!
I haven't done this before but just guessing from what you are trying to accomplish, if these are adherent cells, you can just pipet out the media (making sure not to disturb the cells, should be very easy to do) and run it through some chromotography. Ie: if you know the size, a size exclusion chromotography helps a lot, or if charge, run it through a charged column (anion exchange column). Another quick and dirty way to do it is use a millapore column that seperates by size through centrifugation. Just do a millapore search on yahoo or google.
If these are cells in suspension, you might want to spin down the cells to get a pellet, pipet the supernantant, and then resuspend your cells in fresh culture if you want to continue growing them.
These are just suggestions as I haven't done this before.
As for the comparison in amounts, you might want to try a crude BSA standard curve with coomasie blue stain, or if you have an activity assay, that will work best. A coomasie blue stain detects ALL proteins in your mixture, so you might have to do some controls to normalize background unspecific secreted proteins.
If your protein is secreted from cells, as in, exists in the culture medium instead of staying on the cell surface, it is not necessary for extraction. Just collect the medium and do whatever tests that you need to detect the presence of it, Wb, IP, ELISA... Always take care to have suitable control. Since you want to compare the amount of secreted proteins between transfected and untransfected cells, you will have to make sure that the amount of tested cells are the same. E.g. you can seed the same number of cells into each well, perform tranfection with all necessary controls, and also take care to have untransfected cells. Collect medium from those wells (after suitable time or treatments if those are necessary, e.g. changing medium, time course...) and also collect cells. Medium is for detecting secreted proteins. Cell lysates are for loading control, making sure that you are looking at the secretion of the same amount of cells (so that the possible diffrent in the amount of secreted protein is not due to one sample has more cells than others for example). Also, you can see and compare the amount of the interested protein that still remain inside the cells.
Thank you so much!!!!!!!!!