2 replies to this topic

[Ayah]

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)

[Papaver]

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...

[mdfenko]

Papaver, on 16 July 2012 - 07:13 AM, said:

• 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...

that should read "0.05% sds"