Help for western blot wet transfer blurry bands - (Jul/16/2012 )

Hi please can someone help me with my western blot. I am currently using the western for the BDNF protein and each time I find following wet transfer the marker has 'diffused' across the PVDF. when I develop my bands are blurred and smudged across the lanes.

I preactivate my pvdf in methanol (5 secs) and run the transfer for 60mins at 100V

my transfer buffer is as follows:


8.7g Glycine
17.4g Tris-base
1.11g SDS
600ml Methanol - which i then make up to 3L using distilled water.

I would greatly appreciate any advise anyone has please.

Thank you

(this is my first post - i apologise if i have missed information or it is in the wrong format)

Hey,

so try this:

10X transfer buffer stock
<*>30.3 g Tris base
<*>144.1 g glycine
<*>water to 1 liter

1X transfer buffer - store at 4° C
<*>100 ml 10X stock
<*>700 ml H₂O
<*>200 ml methanol
<*>chill to 4° C
<*>up to 0.5 % SDS can be added if you transfer large proteins...if not, then let it be.

Transfer at 100 V for 1 hour is ok...you can also try 50 min...

Papaver on Mon Jul 16 15:13:41 2012 said: