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Help for western blot wet transfer blurry bands

western blot wet transfer BDNF PVDF membrane

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2 replies to this topic

#1 Ayah

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Posted 16 July 2012 - 06:58 AM

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)

#2 Papaver

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Posted 16 July 2012 - 07:13 AM

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...

#3 mdfenko

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Posted 17 July 2012 - 11:40 AM

  • 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"
talent does what it can
genius does what it must
i do what i get paid to do





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