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Please help with odd bands and smears on Western blot

western blot electrophoresis protein

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4 replies to this topic

#1 argonarin

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Posted 18 October 2012 - 12:24 PM

I am having trouble with my Westerns. We are looking at beta-catenin and using beta-actin as a loading control. My beta-catenin looked smeared. This is the first time this has ever happened. Also some of my beta-actin bands look scrunched up instead of having nice straight bands. Any thoughts?

Attached Thumbnails

  • actin 5 mins small.jpg
  • b-cat 20 mins small.jpg


#2 science noob

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Posted 18 October 2012 - 12:44 PM

1. How did you prepare your cell/tissue lysate?

2. SDS-PAGE running time/settings?

3. Did you block your membrane before primary + secondary staining? With what & how long?

4. Primary/secondary antibody dilutions + incubation times?

5. Did you probe the same membrane twice (first for b catenin and/or b actin followed by b actin and/or b catenin)?

#3 argonarin

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Posted 18 October 2012 - 12:48 PM

1. How did you prepare your cell/tissue lysate?
I lysed BMDC in 75 uL of RIPA buffer with protease and phosphatase inhibitors added. I then froze them at -80.

2. SDS-PAGE running time/settings?
Running time was about 50 minutes on 175 mV.

3. Did you block your membrane before primary + secondary staining? With what & how long?
Membrane was blocked with 5% NFDM for 1 hour. Primary was left overnight, washed 3 times with TBST and then the secondary was added for 1 hour.

4. Primary/secondary antibody dilutions + incubation times?
Primary dilution - 1:1000 for both antibodies overnight at 4 degrees.
Secondary dilution - 1:3000 for one hour at room temp.
These parameters are all suggested by the antibody company (Cell Signaling).

5. Did you probe the same membrane twice (first for b catenin and/or b actin followed by b actin and/or b catenin)?
I probed twice. First for b-cat and then for b-actin. The attached images are of the same membrane.



#4 bob1

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Posted 18 October 2012 - 01:50 PM

It looks like either your gel or running buffer isn't prepared properly. Try remaking the running buffer. You should also use less secondary - the presence of the ladder as a negative band indicates high background, which is usually due to the secondary being at too high a concentration.

Despite the manufacturer's recommendations, antibodies almost always need to be titrated for best use as the signal depends on a whole bunch of factors including the blocking conditions, membrane type, amount of protein loaded, incubation time...

#5 science noob

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Posted 18 October 2012 - 03:21 PM

Yup, I agree with bob1 that secondary antibody dilution can be increased to 1:5000-1:10,000 ( which would explain the background, 'smear' and nagative band in the ladder lane as mentioned my bob1)

The scrunched band seems like a loading/running problem. This normally happens when your pipette tip pokes into the gel when loading, giving it a 'rounded' leading front.
Did you prepare all the samples (all lanes) in the same lysis buffer on the same day?

Did you add a reducing agent (DTT or b-mercaptoethanol) in your cell lysis buffer and boil lysates before running it on the SDS-PAGE?





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