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Peculiar background problem with MPM-2 antibodies


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#1 mayeck

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Posted 19 November 2012 - 12:39 PM

Hi

Couple of days ago I tried my first Western Blotting and the result is pretty ugly. I proceeded with blotting as follows:
- Blocking over the weekend in 5% milk in TBST (with 0,02% sodium azide), 4 degrees with rocking
- 3 hour incubation with mouse monoclonal anti-MPM2 1:1000 (same buffer)
- 1 hour secondary Cell Signalling 1:1000
and developed film with Pierce ECL
Result is shown in picture 1, background is very high and bands from MPM-2 are visible only in one lane.

My reasoning was that 3 days blocking time with milk and tween wasn't very bright idea since MPM-2 detects phophoepitope (and also I've heard, that 4 degrees incubation should be performed without tween). So I dehybridised membrane with standard 0,2 M NaOH and tried to turn whole procedure around, this time:
- Blocking 1 hour at room temperature (milk in TBST, of course no azide here)
- 1 hour goat monoclonal antibody picted somewhat at random from the shelf, established to work earlier by a friend, 1:1000 (Santa Cruz)
- 1 hour Jackson antigoat, 1:10.000
Result (picture 2) seems even worse and the whole blot is very dirty. However the most surprising part is that in this blot bands (although very faint, probably too short primary antibody exposure) can be seen in all 6 lanes.

I'm completely lost at what have happened here and any suggestion should be helpful. Some silly basic mistakes not impossible.

western 01.jpg western 02.jpg

#2 bob1

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Posted 19 November 2012 - 07:43 PM

Milk is full of phospho proteins, you will be better off blocking etc. in BSA or serum based solutions. You should only need to block for 1 hour, but it doesn't hurt (usually) to block for longer.

You may need to titrate the antibodies for best results - try different dilutions, especially for the secondary (assuming you did mean 1:1000 in the first part of your post), which is probably giving you the background in your first image. For 2ries, I ususally use 1:5000 or higher dilution.

Don't lose hope, most people's first WBs look something like that or even just blank...

#3 mdfenko

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Posted 20 November 2012 - 12:25 PM

you don't mention washes between primary and secondary and after secondary. you may need to wash more, as well as changing blocking agent (as bob1 said, there are a lot of phospho proteins in milk).

we never put tween in the block, we want to bind as much blocking agent as possible to reduce background (it may not make much of a difference but it works for us). we use tween in the antibody solutions and washes to reduce non-specific binding.
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