Streaking on nitrocellulose and PVDF membranes

Martin Offterdinger a8803349 at unet.univie.ac.at
Wed Nov 20 10:57:43 EST 1996


On 18 Nov 1996 06:13:07 -0800, tdemeke at pbi.nrc.ca ("Demeke, Tigst")
wrote:

>
>I consistently get streaks on my western blots.  Some of the streaks start 
>from top and continue to the bottom of the gel or stop somewhere in the 
>middle, while others start from the middle or even lower and continue for 
>either long distance or short distance.  I have tried the following: 
>decreasing the amount of protein loaded, reducing the current, lowering the 
>concentration of primary and secondary antibodies, filtering and degassing 
>of acrylamide/bisacrylamide solution, filtering the loading dye, thoroughly 
>cleaning the plates with detergent and water, washing the wells with DDH2O. 
> I still get streaks.  Do you have any suggestions to get rid off the nasty 
>streaks?
>Tigst
>Tdemeke at pbi.nrc.ca
Hi !
1)
How do you prepare your protein samples? Do you disrupt your nuclei
with a strong detergent(SDS) when you isolate your proteins? In this
case you will have a viscous DNA containing protein sample-DNA
fragments can cause a smear on protein gels.You could use a different
detergent to circumvent htis problem and centrifuge off the
nuclei.(Triton X100, Brij 96 etc...)
2) 
How old is the acrylamide solution you use. AA should be prepared
every two month freshly because AA hydrolyses to acrylic acid and than
your gels may exhibit uneven polymerisation causing smearing.

Try to check this two reasons for failure!
Hope this helps, Martin



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