I'm trying to analyze surface membrane proteins from human glial cells.
1. Collect 25 million cells and biotinylate them with 1mg of sulfo-nhs-biotin for 30 min on ice. Buffer is PBS pH 8.
2. Lyse cells with cold RIPA + 2% SDS + PI for 30 min
3. Microcentrifuge at 13,000 RPM for 5 min at 4C. Collect supernatant.
4. Incubate lysate with strep-magnetic beads for 1hr at room temp.
5. Collect beads using magnet stand.
6. Wash 5 times with PBS.
Then I run SDS-PAGE in-gel digestion with trypsin and prep sample for mass spec. analysis. This part of the experiment is following a well established protocol used in the lab.
I'm only getting about 700 identified proteins and only 7-10% are transmembrane proteins and 60-70 are cytoplasmic proteins.
So why do you think my transmembrane yield is so low?
I should mention that my biotin is dissolved in DMSO and final concentration in cell during biotinylation step is 1%. (I highly suspect this conc. of DMSO is causing internalization of biotin)
What do you think?
Any input will be great.
Thank you
The 1% DMSO may increase the permeability of cells to sulfo-NHS-biotin, as you suggested. In addition, since you did not quench the reaction prior to the addition of RIPA buffer, some labeling of cytoplasmic proteins may have occurred during and after cell lysis.
Thank you Adam Shapiro! I forgot to mention that I do quench excess biotin with PBS + 100mM glycine.
Another possibility I was thinking of was the viability of cells at the beginning of my experiment.
Is it possible that if I begin with some dead cells (maybe around 25% of cells are dead) that would allow biotin to bind to cytoplasmic proteins of dead cells? I do three washes with cold PBS after collecting cells to get rid of medium. Wouldn't these washed get rid of dead cells and proteins from them?
I'm just trying to think of everything that could lead to my issue.
Is the cell viability measured by trypan blue exclusion and so you have microscopic visualization of the cells? How do the cells appear microscopically just before biotinylation? Indeed, DMSO must be cold when added to cells for cyropreservation purposes. The reaction was conducted with cells on ice but was the biotin/MSO solution near 0C when added to the chilled cells? A transient warming may have affected the cells.
Thank you Grant Shimamoto
Yes, cell viability is checked with Trypan blue visually under the microscope. Visually, there isn't a lot of debris but I do see some dead cells about 28-30%. Live cells (those that are not blue) look round and happy.
Yes, biotin in DMSO was always on ice before addition to the cells.
So today I checked to see if biotin in 1% final conc. DMSO will cause cells to lyse. I collected cells just as I would followed up washes and 30 min incubation of biotin in 1% DMSO. I checked with trypan blue before biotin incubation and after biotin incubation.
Cells looked healthy (no blue cells).
Although cells may be viable but membrane is permeabilized to allow biotin internalization...
In the end, I'm ordering new no-weigh formula biotins simply because I cannot trust this biotin in DMSO.
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