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Help! Half membrane exposed perfectly. Half membrane is cloudy


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#1 B.B.

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Posted 20 May 2011 - 04:33 AM

Hello,

I'm new to this forum and also to Western Blots.
I've been following a protocol given to me by a post doc and am having a little trouble with exposure.
Here is the protocol in breif:

Cells are grown and harvested with lysis buffer. The sample is homegenised, spun down and collected.
The samples are run on a lab-made 10% gel and transfer is done on ice at 100V.
I check for protein transfer for Ponceau Red (which was positive) and I cut my membrane according to MW (referring to the ladder) so that I am able to stain for two proteins (housekeeping proteins and protein of interest).
The membrane (nitrocellulose) is blocked in 5% milk for 1 hour at room temp.
The first antobody (made in 5% milk) is incubated on a rocker at 4*C overnight.
The second antibody (also made in 5% milk) is incubated on a rocker at room temperature for 1 hour.
All washes are done with TBST. I aim for at least 5 washes for 5 minutes for each membrane.
I use West Pico Pierce Chemiluminiscent for developing.

The problem I am experiencing is that one half of the membrane (with the protein of interest on it) is exposing perfectly. The other half (with the housekeeping antibodies) is messy, unspecific and prdocuing a black outline on the membrane.

As I treat these two half of the membranes exactly the same, I can only think it's the antibodies I use for the housekeeping protein.

The concentrations I ues are:
HOUSEKEEPING PRIMARY 1:500, SECONDARY 1:1000
PROTEIN OF INTEREST PRIMARY 1:4000 SECONDARY 1:1000

Has anyone else had this very unusual problem? Or thinks they may be able to shed some light on it!

Bev

#2 casandra

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Posted 20 May 2011 - 05:12 AM

Hello,

I'm new to this forum and also to Western Blots.
I've been following a protocol given to me by a post doc and am having a little trouble with exposure.
Here is the protocol in breif:

Cells are grown and harvested with lysis buffer. The sample is homegenised, spun down and collected.
The samples are run on a lab-made 10% gel and transfer is done on ice at 100V.
I check for protein transfer for Ponceau Red (which was positive) and I cut my membrane according to MW (referring to the ladder) so that I am able to stain for two proteins (housekeeping proteins and protein of interest).
The membrane (nitrocellulose) is blocked in 5% milk for 1 hour at room temp.
The first antobody (made in 5% milk) is incubated on a rocker at 4*C overnight.
The second antibody (also made in 5% milk) is incubated on a rocker at room temperature for 1 hour.
All washes are done with TBST. I aim for at least 5 washes for 5 minutes for each membrane.
I use West Pico Pierce Chemiluminiscent for developing.

The problem I am experiencing is that one half of the membrane (with the protein of interest on it) is exposing perfectly. The other half (with the housekeeping antibodies) is messy, unspecific and prdocuing a black outline on the membrane.

As I treat these two half of the membranes exactly the same, I can only think it's the antibodies I use for the housekeeping protein.

The concentrations I ues are:
HOUSEKEEPING PRIMARY 1:500, SECONDARY 1:1000
PROTEIN OF INTEREST PRIMARY 1:4000 SECONDARY 1:1000

Has anyone else had this very unusual problem? Or thinks they may be able to shed some light on it!

Bev

Hi Bev...welcome to Bioforum..... experiencing the newbie western blues,eh....:)

-try incubating the housekeeping primary (actin, tubulin?) just for 1 or 2 hours at room temp...

-you may also wanna try increasing its dilution...actually, even your secondary Ab dilution can also be increased to 1:5000 or 1:10000 as what most standard protocols/manufacturers recommend....

-and also, if you're gonna use the housekeeping protein to normalise or as a loading control, then you have to strip the membrane that you've already probed for your protein of interest then re-blot it using your housekeeping protein Ab so you don't need two separate membranes for this unless you're just testing the housekeeping protein Ab for the first time....but surely, someone has been using it before and it hopefully works, eh?

Edited by casandra, 20 May 2011 - 05:13 AM.

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#3 B.B.

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Posted 29 June 2011 - 04:40 AM

Hi,
thanks for the reply.
It turns out that my secondary antibody for my housekeeping protein was dud!
I had been given an aliquot of it from another post-doc, which I stored in the freezer. Turns out it can only be stored at 4*C! Whoops! So when I defrosted it to use on another Western Blot, my blots were fuzzy and messy.
Never mind - lesson learnt. And at least it was only a small aliquot :)
Bev




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