Methods and Techniques Discussion => Western Blotting (WB) => Topic started by: nourani on December 02, 2007, 03:03:23 AM

Title: How many sample i need for western blot
Post by: nourani 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. 
Title: Re: How many sample i need for western blot
Post by: ImmunoNYC 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.
Title: Re: How many sample i need for western blot
Post by: ImmunoNYC 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.
Title: Re: How many sample i need for western blot
Post by: Cardio 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.