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antibody works in IHC but not on blot?

antibody works in IHC but not on blot? - Western Blot Forum

antibody works in IHC but not on blot? - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 07-21-2009, 02:01 PM
Pipette Filler
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Default antibody works in IHC but not on blot?



Hello,

I am doing a lot of IHC (immuno histo chem). I have a long list of antibodies which have worked in IHC and I now have to do westerns to demonstrate antibody specificity.

I have begun by testing my rabbit anti-GFAP antibody. I have done a blot in duplicate (5 wells the same on the SDS-PAGE). For a positive and loading control I am using mouse anti-a-tubulin. I have used protein samples (all extracted in RIPA) of rat muscle (which should be GFAP negative), zebrafish brain, goldfish brain (the antibody has worked on both previously in Western Blots) and the test samples (Nothobranchius sp. brain).

On the one half of the blot I only added anti-GFAP. On the other half I added anti-GFAP and anti-a-tubulin. Then I incubated with avadin linked HRP anti-rabbit and anti-mouse antibodies. The secondaries were incubated together. Blocking solution was TBS + tween with 1% milk powder.

I did not get a GFAP positive band on either blot but I did get a a-tublin positive band in all lanes on the blot for which it was probed.

For the IHC I am using PBS.

I am re-running the gel and plan to proceed by trying the following:

1. Using TBS without tween to reduce the specificity. The GFAP protein was of mouse origin and I am working with a fish, Nothobranchius sp.

2. Can I blot in PBS? I have in the past had trouble using the antibodies using TBS as the buffer but others haven't.

Any advice or suggestions would be much appreciated.

Thanks
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Old 07-30-2009, 03:50 PM
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Default Re: antibody works in IHC but not on blot?

How much protein have you loaded per well?
I've done a lot of westerns and recently been doing a lot of IHC. Some antibodies for IHC don't always work well for Westerns, and vice vera. You may need more protein loaded per well to get a signal on a blot for some antibodies- usually 20-30 µg will do for most antibodies, but I have had samples where up to 50-60µg of sample protein are required to get a signal with a western
Also, there is the issue of the sample prep/storage and the sample pretreatment can affect the protein expression for westerns...some proteins degrade quickly and require special care. You can extract protein for Westerns from parrafin embedded tissues, but frozen/fresh tissue is always preferred.

I'd recommend increasing your protein per well and see if you get a signal.
Just a thought
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Old 08-03-2009, 09:01 AM
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Default Re: antibody works in IHC but not on blot?

Hello,

Thanks for the reply Mad Scientist but the problem is sorted. I switched from TBS (pH 7.2) with 0.05% tween to PBS (pH 7.6) with 0.05% tween using 5% milk powder to block. It may be a pH problem rather than buffer but it works (for now) so I am happy.

[Protein] was not the problem. What bands I got using actin as + control were big and fat (I actually had to reduce the amount of protein I was loading to get sharp bands).

Thanks again
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Old 07-05-2013, 02:12 PM
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Default Re: antibody works in IHC but not on blot?

Hi, tyronegenade

What salt do you use to prepare PBS?

Tks
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Old 07-05-2013, 02:40 PM
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Default Re: antibody works in IHC but not on blot?

Hi, tyronegenade

What salt do you use to prepare PBS? How can I know if the antibodies that I have doesn´t work to blot?

Tks
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Old 07-06-2013, 09:49 AM
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Default Re: antibody works in IHC but not on blot?

Hello,

My blots now work perfectly.

This is my PBS recipe:
148 mM Na+, 4.17 mM K+

1 L 1×
0.14 M NaCℓ → 8.19 g NaCℓ
2.7 mM KCℓ → 0.2 g KCℓ
8.8 mM Na2HPO4 → 1.25 g Na2HPO4 (Mr = 141.96 g/mol)
1.47 mM KH2PO4 → 0.2 g KH2PO4 (Mr = 136.09 g/mol)
Adjust pH to 7.4
Fill to 1 L with mQH2O
Store at room temperature

0.5 L 10×
41 g NaCℓ
1.0 g KCℓ
6.3 g Na2HPO4 (Mr = 141.96 g/mol)
1.0 g KH2PO4 (Mr = 136.09 g/mol)
Adjust pH to 6.74, when diluted to 1x it should be pH 7.4.
Fill to 500 mL with mQH2O
Store at room temperature

For the blots I use PBS-tween (500 microL tween per L).

Priscilla, what antibodies are you working with and how are the proteins extracted.

If the antibody you are using hasn't been tested on western blots then you have to try the antibody at several dilutions. Generally, if you use 1:100 for IHC, then you can start blotting with 1:1000. I would try 1:500; 1:1000 and 1:2000.

The milk blocking solution might contain the very protein you are looking for so it may be a good idea to block in 5% milk powder and then rinse with PBS tween before adding the antibody.

I hope this helps.
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Old 07-12-2013, 11:46 AM
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Default Re: antibody works in IHC but not on blot?

Hi, tyronegenade

I tried blot this week. But, I didn´t have sucess. My samples are intestine from rats. The protein that I´m testing is GFAP.

This is my protocol.
Proteins (30 μg/well) were separated on a 10 % gel ( deionized water, Bisacril 30%, Tris HCl 1,5M pH 8,8, SDS 10%, PSA 10% , TEMED).

I ran in 80V after about 20 minutes I switched to 110V.

I let it the nitrocelluse membrana and the filter paper soaked in buffer transfer (Tris base, glycine, methanol and deionized water).

P S: I made two buffer transfer, one with SDS the second without SDS.

I transferred to nitrocellulose membranes at 25 mv per 40 minutes. (semi-dry transfer) (Biorad).

The membranes were blocked with 5% non-fat milk in TBS with 0.1% Tween-20 for 1 hour at room RT,

Washed 2 times (5 min) with TBS-T,

Incubated overnight in TBS-T at RT with monoclonal GFAP produced in mouse ( IgG1 isotype) (1:500).

After being washed in TBS-T (3×5 minutes), the blots were incubated for 1 hour at RT with the appropriate HRP-conjugated secondary
antibody (1:1000).

The membrane that I used buffer transfer with SDS. I didn´t have bands.

The membrane that I used buffer transfer with no SDS I got a weak bands.

What Do I do wrong? Can you help me? Do you think that the problem is the TBS?

Thanks a lot.
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Old 07-15-2013, 09:07 AM
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Default Re: antibody works in IHC but not on blot?

Hello,

Go have a look here: [Only registered users see links. ] While I see some evidence in Pubmed for GFAP expression in the intestine I doubt it is expressed in large amounts. You may not have loaded sufficient protein to see any GFAP. But with 30 ug already loaded you already have a lot of protein in the gel... You could require a more sensitive detection method.

I use the GA5 and DAKO Z0334 anti-GFAP antibodies. These work well. They have very low titer values and are very sensitive. Which antibody are you using?

I also use PBS and not TBS. I switched to PBS because I wasn't getting good binding with the TBS. You don't need to reblot, just go back and reincubate the blot with antibody in PBS. Try several antibody dilutions...

You secondary dilution of 1:1000 may be too low. I use secondaries at 1:3000. At lower dilutions you could get steric hinderence of the binding of the secondary.

I don't use SDS in my transfer buffer. There is no need for it.

A last question: what buffer are you using for extraction? I use RIPA with deoxycholate and high SDS to break the protein-protein interactions. GFAP is an intermediary filament and partakes in strong protein-protein interactions. If you are using a buffer with weak protein-protein extraction potential then most of the GFAP may precipitate out of solution.
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