Hi everyone,
I am trying to fractionate my C2C12 myoblast cells using a protocol for cellular differentiation I found online:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678309/ but for some reason using 1 T75 flask with the cells being 85% confluent didn't give me enough protein in any of the fractions to perform western blotting, I repeated the experiment using 3 T75 flasks containing cells once again that were about 80-85% confluent and still did not get enough protein in any of the fractions to amount enough to make western blotting samples, however there is some protein in there.
I'm not sure what I should change about the protocol, I was thinking maybe 400ul of each lysis buffer is too much and to decrease the volume of that in order to increase the amount of protein found in the supernant fractions or I was wondering if maybe swelling the cells using a hypotonic buffer would be useful?
I am specifically looking for the plasma membrane, mitochondria and nuclear fractions, if you have any protocols to differentiate C2C12 cells other than this protocol I found please share them with me.
I appreciate any feedback you may have.
Here is the protocol steps just incase the link doesn't work:
1. Remove culture medium and wash the cells with room temperature phosphate buffered saline solution.
2. Trypsinize the cells by adding 800 μL of 0.25% Trypsin: 0.9 mM EDTA: phenol red solution (Gibco Life Technologies, 25200) and incubating the cells at 37 °C for 2 min or until cells are detached.
3. Add 5 mL of culture medium containing 10% fetal bovine serum to inhibit trypsin activity, and collect the cells.
4. Centrifuge at 500 × g for 10 min at 4 °C to pellet the cells.
5. Using a 1 mL pipette and tip, discard the supernatant and resuspend the pellet by pipetting up and down in 500 μL of ice cold PBS.
6. Centrifuge for at 500 × g for 10 min at 4 °C to pellet the cells.
7. Discard the supernatant and add 400 μL of ice cold lysis buffer A supplemented with 4 μL of protease inhibitor cocktail.
8. Incubate on end-over-end rotator for 10 min at 4 °C.
9. Centrifuge at 2000 × g for 10 min at 4 °C.
10. Collect the supernatant. This fraction contains the cytosolic proteins.
11. Add 400 μL of ice cold lysis buffer B supplemented with 4 μL of protease inhibitor cocktail and resuspend the pellet by vortexing.
12. Incubate on ice (or at 4 °C) for 30 min.
13. Centrifuge at 7000 × g for 10 min at 4 °C.
14. Collect the supernatant. This fraction contains the proteins from membrane-bound organelles (mitochondria, endoplasmic reticulum, Golgi, etc.) except those from the nucleus.
15. Add 400 μL of ice cold lysis buffer C containing 7 μL of Benzonase and 4 μL of protease inhibitor cocktail.
16. Incubate on an end-over-end rotator for 30 min at 4 °C to allow complete solubilization of nuclei and digestion of genomic DNA.
17. Centrifuge at 7800 × g for 10 min at 4 °C.
18. Collect the supernatant. This fraction contains the nuclear proteins.
19. Pellet contains nuclear proteins and protein complexes that resist extraction and typically include active RNA polymerases and regulatory proteins. These can be solubilized with lysis buffer C supplemented with 8 M urea for analysis, or discarded.
I imagine you are doing a step wrong as even with a single T175 flask you should obtain lots of protein.
Are you sure all your cells are detaching? Check you flask after trypsinising under the microscope. Do you have a large cell pellet to start with?
I would also check your calculations for the reagents (lysis buffer etc) that you made and make them fresh when you repeat.
Swelling your cells won't help as the proteins are being released from a detergent that permeabilises the membranes. You are not looking to burst you cells. You only need to swell your cells if you are using mechanical lysis. The first low concentration detergent will permeablise the plasma membrane releasing cytoplasmic proteins. The second lysis step solubilises the organelles just leaving the nucleus which is harder to break down.
There is a more recent paper following a similar method "Mass Spectrometry Compatible Subcellular Fractionation for Proteomics", it might be worth a try.
Alternative methods might be differential centrifugation rather than detergent-based methods. Look up Lamond lab protocols.
Try to precipitate the proteins first from their respective supernatants by an appropriate method e.g. acetone or CHCl3 or methanol precipitation. Re-suspend in an appropriate buffer.
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