Western blot sample loading question - (Mar/27/2012 )

I dont know the concentration of my samples so I tried a few dilutions to run on a gel.
I ran 1:10, and 1:100 dilution. I could see a band at 1:10 but not 1:100.
I want to try running samples at 1:2 or neat.
How do I go about making up the samples in loading buffer for loading onto the gel.
I am using 2x sample loading buffer.

When I did 1:10 and 1:100 dilution previously I added 45ul of 2X sample loading buffer and 5ul sample. Then made a 1:10 dilution of this to get 1:100 dilution. I used 2X loading buffer again to do this dilution.

This time I was thinking of doing 10ul sample + 10ul 2X loading buffer to get 1:2 dilution. How do I go about loading the neat sample? Do I need to add the loading buffer at all? The sample is already in lamelli buffer?

I am now thinking that I didnt load the 1:10 and 1:100 correctly. I should have had 50% 2X loading buffer and 50% 1:10 sample. Is this correct?

How do people normally load samples when dealing with dilutions?

If the sample is already in Laemmli buffer, then there is no need to add additional loading buffer as it already contains the glycerol and salts necessary for running, unless you are making a dilution (though why you would is beyond me, you could just load a smaller amount...). For protein quantitation people typically do a plate based method, such as the BCA or Bradford assays.

To make the dilutions you are correct in your first example of a 1:10 dilution.