How to lyse animal cell line? - (Dec/30/2007 )
Hi,
I would like to ask what is the suitable lysis buffer for lysis of animal cell line.
I want to lyse B16F10 murine skin melanoma cells to release the tyrosinase.
I would like to ask what is the suitable lysis buffer for lysis of animal cell line.
I want to lyse B16F10 murine skin melanoma cells to release the tyrosinase.
Assay buffer with 0.1% Triton X100.
i used to lyse cell to count bacteria invaded into cells aft infection -- i use 0.1% triton X 100 in PBS.
When i need to extract RNA out of my cells, I just resuspend cells with TRI reagent, pipette up and down >30times... it lysed.
depends on what the is the purpose of you to lyse your cells.
I would like to ask what is the suitable lysis buffer for lysis of animal cell line.
I want to lyse B16F10 murine skin melanoma cells to release the tyrosinase.
Assay buffer with 0.1% Triton X100.
Thanks for your reply. But when i go through some research journals, the researchers used phosphate buffer (pH 6.8) containing 1% Triton X-100 together with 0.1 mM phenylmethylsulfonyl fluoride (PMSF). Why need to used so high percentage of Triton X-100? Is it necessary to add in PMSF (a protease inhibitor) in the lysis buffer?
When i need to extract RNA out of my cells, I just resuspend cells with TRI reagent, pipette up and down >30times... it lysed.
depends on what the is the purpose of you to lyse your cells.
The lysis buffer should be sterilised as well before adding into the cells?
The purpose I lyse the B16F10 murine melanoma cells is to release the tyrosinase (a transmembrane protein). I want to study the tyrosinase inhibition activity when test with diffferent plant extracts.
0.1 % is enough for in plate lysis and assay, which I assume you will do. The amount of triton X100 should be proportional to the amount of cells. Maybe that work used cell pellet therefore more triton was used.
People use a cocktail of protease inhibitors. you may do so as well. These can be purchased directly.
You dont need to sterilize the triton lysis buffer. Just keep it at 4 C.
Higher triton is used when some protiens are linked to membranes and to extract them efficiently, you will need higher concentrations of detergents. But for your samples, I don't think you will need high concentrations.
People use a cocktail of protease inhibitors. you may do so as well. These can be purchased directly.
You dont need to sterilize the triton lysis buffer. Just keep it at 4 C.
If I plan to run the assay in 96-wells microplate, I just need to used 0.1 % triton X-100 lysis buffer for each well in order to extract the tyrosinase transmembrane protein from the murine B16F10 skin melanoma cells?
How do I determine the optimal number of cells loaded per well in this case?
After add in the Triton X-100 lysis buffer, what lysis method should I perform? Such as freeze-thaw lysis ?
People use a cocktail of protease inhibitors. you may do so as well. These can be purchased directly.
You dont need to sterilize the triton lysis buffer. Just keep it at 4 C.
If I plan to run the assay in 96-wells microplate, I just need to used 0.1 % triton X-100 lysis buffer for each well in order to extract the tyrosinase transmembrane protein from the murine B16F10 skin melanoma cells?
How do I determine the optimal number of cells loaded per well in this case?
After add in the Triton X-100 lysis buffer, what lysis method should I perform? Such as freeze-thaw lysis ?
We can not decide it for you. It has something to do with the enzyme level in cells and the product optical property.
Be aware that this line of cell changes its tyrosinase level fairly fast within a few passages. The enzyme level is not fixed.
You just need to incubate the cells in lysis buffer for 5 min at RT or 37 C. Cells get lyzed and enzyme leaks out.
In fact, I think you can add the substrate to the lysis buffer and do the lysis/assay together.
We can not decide it for you. It has something to do with the enzyme level in cells and the product optical property.
Be aware that this line of cell changes its tyrosinase level fairly fast within a few passages. The enzyme level is not fixed.
You just need to incubate the cells in lysis buffer for 5 min at RT or 37 C. Cells get lyzed and enzyme leaks out.
In fact, I think you can add the substrate to the lysis buffer and do the lysis/assay together.
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Really appreciate ur kindness reply.
When cells get lyzed and enzyme leak out, shall I clarified the cellular extracts by centrifugation at 10,000 rpm for 5 min at 4 degree celcius? The tyrosinase will be present in the supernatant and I just take the supernatant and add with substate to run the assay. What do you think?