How to prepare blood serum for western blot? - (Sep/19/2012 )

Hi!
I found a protocol from
http://www.protocol-online.org/biology-forums/posts/32178.html

For human serum I obtained 77 mg/mL

What is final concentration after mixed blood serum with Laemmli buffer?

I'm normally working with cell pellets to run WB and I got 1 mg/mL for final concentration to get good band for my protein.

-Tai-

The concentration is any concentration you choose - and should be optimized depending on the abundance of the protein you are looking for. I would recommend choosing a starting point (say 10 mg/ml) and working down from there in a serial dilution to get a level where you can see nice clean bands.

-bob1-

thank you very much for your advice

-Tai-

Hi
here is my first try WB with blood serum.

http://imageshack.us/photo/my-images/69/membraneh.jpg/
From picture above,the first 4 lanes are normal sample from the whole cell extraction, in lane 5-8 are my first test blood serum.

Total protein I loaded is 10 ug (1ug/uL) in all samples. For the last 2 is my positive control.

Could someone give me some advice if what is commonly use for internal standard. I normaly use beta actin 43kDa but in the srerum sample, It seems not work.

Albumin seems to be too high, or not ?, because I'm plaining to increase total protein concentration for the next time.

-Tai-