We biotinylate live cells (adherent monolayer of Ishikawa) for 1 hr at 37C. This is followed by quenching and washing with PBS. However after scrapping in PBS, followed by centrifugation at 2000 -3000 rpm for 15-20 min, cells do not settle down. It takes almost 30 min at 5000rpm to see a thin pellet. This is not the case with nonbiotinylated cells treated in the same fashion. Does biotin labeling alter buoyancy of cells? Is it ok to spin at 10,000 rpm or will it lead to removal of biotin tag from proteins?
Well, if you do not need living cells at the end, that would be fine for your proteins.
I think you are lysing cells with the biotinylation procedure and therefore getting a smaller pellet. Try doing the biotinylation for 30 min-1 hr with the cells on ice.
If you are using an amine reactive biotinylating reagent you will not lose it from your protein, unless you do not have proteinase inhibitors in your lysis buffer.
I wonder if you are killing the cells while scraping them. Usually surface biotinylation is very strong (amide reaction) and will not fall off and it shouldn't alter the buoyancy of the cells. It is not ok to spin cells at high speeds if you want live cells at the end of the biotinylation process. Try adding 0.1% BSA to your PBS for the washing step, may be the cells are sticking the polystyrene tube during the spinning down process.
Thanks Stefansson and Bhuvana... We are biotinylating the cells to obtain the cell surface proteins. This will be followed by the enrichment of biotinylated proteins using streptavidin agarose beads and then immunoblot with specific antibodies. However our worry is the poor recovery of total protein (biotinylated as well as nonbiotinylated). In your experience, how much is the recovery of cell surface proteins (in ugs) from 10 million cells?
We use the biotinylated proteins for western blot. We opt to do whole cell lysate just after washing, to avoid this situation. The lysates will be cleared by centrifuge and equal amount of total protein was pull down with streptavidin beads, eluted with 2x sample loading buffer, and then run western blot. It is basically an IP procedure after washings. It works pretty well for us.
Have mercy on those poor cells! Try 300 rpm for 5 minutes. If your supernatant isn't nice and clear with a fluffy cell layer at the bottom, then you can try either a little faster or longer. If your centrifuge doesn't have settings that low-- there's the problem.
Are you working ice cold? Cold cells are more fragile. Also, can we assume using standard 15 ml plastic conical tubes, not eppendorfs into a microfuge?
In any event, those speeds and times would puree the cells.
Hi Geetanjali. I think the most likely cause of your problem is that the biotinylating reagent or the solvent it is in, is killing your cells.
Keeping the cells for 1 hr at RT in the pesence of something that is toxic to them will cause cell lysis and loss of protein. Scraping the weakened cells afterwards will make things worse.
I think Jiang has the best solution.
I worked with non-adherent cells ( B cells). We used 10 million per tube too. We lysed them at 10million/ml concentration. Unfortunately, because of an issue with our lysis buffer and the protein assay kit we had, we never measured the protein recovered with a kit. We normalized the blot with both cell equivalents count (count before lysing) and gapdh or beta actin. I know it is crude but that is what we did. So, I cannot give you the protein recovery in micrograms. We did lyse the cells immediately after the wash (like Jiang) and we too either pulled down all the surface protein with streptavidin and blotted for specific proteins on the surface or probed with streptavidin-HRP to see all the surface proteins that came up. I looked at BCR and its associated molecules.
I had 10 million/condition. Ran a million cell equivalents on the gel/well and got very good recovery. I had to develop them for mere seconds to get wonderful blots.
Yoo have not provided enough infor for me to help with out just giving you a protocol.
what type of buffer are you using for biotinylation and the type of biotin...
What are you using to quench the biotin...glycine or TBS?
Why are you doing it at 37 degrees? 4 degrees (on ice) for 30 minutes is fine as long as there are sufficient lysine residues on your surface molecule.
Secondly, why not lyse the cells directly from the plate instead of lifting and or scraping?
please check this pub for a detailed protocol PMID: 22298529
I recommend doing the biotinylation on ice (30min). You can add 0.1mM CaCl2, 1mM MgCl2 to the PBS to avoid detaching of the cells during the incubation.
It appears that you over-biotinylated and killed the cells so that you are dealing with cell "ghosts" that lost their buoyant density. I would recommend to lower biotin input and slow the reaction by cooling.
Thanks Bernhard... makes sense... we will try biotinylation at 4 degree...
What can be used as a loading control for western blotting to normalize the levels of total protein that have been biotinylated?
As long as it is expressed on all nucleated cells at equal levels which is the case for MHC class I and beta2M
Also depends on the tubes you used. Polystyrene or polypropylene. STyrene might work better
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