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Fixing concentration for western blot from MTT


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#1 Sathiya Sekar

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Posted 27 October 2013 - 10:46 PM

Hi,

Why should we use 96 well plate for cytotoxicity and 6 well plate for western blot assays. For example, for MTT assay we used 96 well plate (8,000 cells/well) and obtained an EC50 as 20mcg. Now how to fix the concentrations for western blot assays? Moreover the total number of cells we used in 96 wells plate will be 8000 cells/well, but for 6 well plate we will use 1X105 cells. Is that concentration and number of cells wont correlate each other?

Thank you

Sathiya   



#2 bob1

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Posted 29 October 2013 - 12:14 PM

Your "concentration" of 20 mcg (micrograms? usually abbreviated to ug?)  is not a concentration - that is an amount.  A concentration is something like 20 ug/ml and should work irrespective of the volume as the volume is accounted for in the concentration.

 

You need to use the 6 well plate so that you have enough cells to detect your protein - it would be very difficult to do this from one well of a 96 well plate and very time consuming to harvest many wells from a 96 well plate.

 

You may find that the activity of your drug is dependent on cell density as well as concentration.






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