Author Topic: How many sample i need for western blot  (Read 7585 times)

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Offline nourani

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How many sample i need for western blot
« on: December 02, 2007, 03:03:23 AM »
Hi All
I would like to detect a protein via Wester blot from Lung tissue( TBLB
) . please let me know that  how many sample ?/mg i should obtain. 

How many sample i need for western blot
« on: December 02, 2007, 03:03:23 AM »

Offline ImmunoNYC

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Re: How many sample i need for western blot
« Reply #1 on: November 04, 2008, 08:51:52 PM »
This is impossible to answer. Generally it is presumed western can detect picograms of protein. However depending on the expression level of your protein, you might need loads or little of your lysate. I suggest you load as much as possible and do a titration for your first experiment: 200, 100, 50, 25, 12, 6 ug of total lysate.

Offline ImmunoNYC

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Re: How many sample i need for western blot
« Reply #2 on: November 04, 2008, 08:53:13 PM »
Also note that 200-250 ug is roughly the most you should load or your gel might start to run funny.

Offline Cardio

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Re: How many sample i need for western blot
« Reply #3 on: November 11, 2008, 02:48:47 PM »
Just to add to NYC. If you are old school and poor your own gels then I would run a large gel over night and flank the sides with loading dye. This will help prevent the smiley face effect that NYC mentioned and compress the lanes so its easier to read.

IF you know that you protein of intrest is expressed in the lung in relative large amounts then 100 microgram is plenty to detect it. Just go on Pubmed and do some research on other publications. 

Re: How many sample i need for western blot
« Reply #3 on: November 11, 2008, 02:48:47 PM »