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Dot-blot with immobilized ABs on membrane for protein detection


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8 replies to this topic

#1 Fribytter

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Posted 05 July 2011 - 06:36 AM

Hi all,

I have some trouble by thinking of an experiment where I would like to make a dot blot, but not a usual one with antigens attached on the membrane, but the other way around.

I would like to attach antibodies to the membrane (preferably both primary and secondary), so I could have a ready-made membrane one could "soak" in to a cell lysate and detect proteins in there. Does anybody of you know whether this would be possible? The probed membrane should off course be proceeded with some ELC (or like) to detect the signal.

Any suggestions?

Thanks in advance

#2 knuf

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Posted 05 July 2011 - 06:45 AM

I don't think this will work because if your primary antibody is already attached to the membrane then you're secondary will bind and thus, you will get a signal regardless of whether or not the antigen is present in the lysate. I don't see how you could make this antigen-dependent.

#3 Fribytter

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Posted 05 July 2011 - 06:48 AM

I don't think this will work because if your primary antibody is already attached to the membrane then you're secondary will bind and thus, you will get a signal regardless of whether or not the antigen is present in the lysate. I don't see how you could make this antigen-dependent.



Thanks for your reply.

I was thinking, that if the sequence of the immobilization was to incubate with secondary ABs on the membrane, wash, incubate with primary ABs, wash, and then with cell lysates (antigens). Would e.g. ECL bind to the HRP conjugate on the secondary AB then?

#4 little mouse

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Posted 05 July 2011 - 07:15 AM

what you want to do looks like the pregnancy test.
on one side (lets say the right one) you have attached Ab.
on the other side (left) you have mobile Ab coupled to a dye. this is the side where you put your sample too, then everything migrate to the other side, the antigen bounded to the mobile antibody binds to the attached Ab and you see a colored line due to the accumulation of the dyed antibody.
have a look on internet, I'm quite sure you will find plenty of webpages explaining it.
Now, how to do it... good luck

#5 Fribytter

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Posted 05 July 2011 - 07:30 AM

what you want to do looks like the pregnancy test.
on one side (lets say the right one) you have attached Ab.
on the other side (left) you have mobile Ab coupled to a dye. this is the side where you put your sample too, then everything migrate to the other side, the antigen bounded to the mobile antibody binds to the attached Ab and you see a colored line due to the accumulation of the dyed antibody.
have a look on internet, I'm quite sure you will find plenty of webpages explaining it.
Now, how to do it... good luck


It does not have to be double test like a pregnancy test, rather a semi-quantitive dot blot which is fast to achieve, with many different cell lysates, and get fast reliable results.

I know it has been done before, and some companies are offering ready-made membranes to test different proteins from cell lysates, like here for example: http://www.rndsystem.../pdf/ARY018.pdf

but how do they do it?

#6 almost a doctor

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Posted 06 July 2011 - 01:19 AM

Hi, as kfunk said I don’t think what you are trying to do will ever work. If your secondary and primary are already in the membrane, the signal is always going to come back positive. In fact the idea doesnt even make sense, think about it. The secondary antibody is your detection antibody and will only give signal if the primary has bound to the target. As I said initially, if the antibody is already there, there’s always going to be signal.

The protocol in the link you posted basically works as a sandwich ELISA. The ONLY antibody initially bound to the membrane is a capture antibody (or primary antibody 1, which will bind your target). Then you mix your sample with detection antibody, which will also bind to the target antigen (we could call it “primary antibody 2” to distinguish it from your typical secondary antibody). The target antigen will bind both the detection antibody (biotinylated), and the capture antibody in the membrane. Is this sandwich system that makes the whole assay specific. After this, you add streptavidin-HRP which will bind to the biotin in the detection antibody and only the spots with your target will light up.

Hope this makes sense.

#7 Fribytter

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Posted 06 July 2011 - 04:42 AM

Hi, as kfunk said I don’t think what you are trying to do will ever work. If your secondary and primary are already in the membrane, the signal is always going to come back positive. In fact the idea doesnt even make sense, think about it. The secondary antibody is your detection antibody and will only give signal if the primary has bound to the target. As I said initially, if the antibody is already there, there’s always going to be signal.

The protocol in the link you posted basically works as a sandwich ELISA. The ONLY antibody initially bound to the membrane is a capture antibody (or primary antibody 1, which will bind your target). Then you mix your sample with detection antibody, which will also bind to the target antigen (we could call it “primary antibody 2” to distinguish it from your typical secondary antibody). The target antigen will bind both the detection antibody (biotinylated), and the capture antibody in the membrane. Is this sandwich system that makes the whole assay specific. After this, you add streptavidin-HRP which will bind to the biotin in the detection antibody and only the spots with your target will light up.

Hope this makes sense.


Hi there,

youre absolutely right, that all secondary AB will be bound to the membrane, and will give signal no matter what. It is, however, the sandwich ELISA kind of membrane I would like to make. I was just wondering about some few things:

- in the above description one will need two different primary antibodies recognizing the same antigens, right?

- When the membrane (where the first primary AB is bound) is exposed to a cell lysate (i.e. the antigens), then the "soup" with the second primary ABs should be added; how do you make sure that the different antibodies do not cross-bind?

Do you happen to know any reference using/describing this method. I have tried to search, but I really dont know which "buzzwords" to use in the search engines.

thanks..

#8 almost a doctor

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Posted 06 July 2011 - 08:28 AM

- in the above description one will need two different primary antibodies recognizing the same antigens, right?

YES

- When the membrane (where the first primary AB is bound) is exposed to a cell lysate (i.e. the antigens), then the "soup" with the second
primary ABs should be added; how do you make sure that the different antibodies do not cross-bind?


As far as I know for sandwich ELISA two different antibodies against the same protein are used. This can either be a polyclonal and a monoclonal antibody, or two different monoclonal antibodies that recognise different epitopes of the protein of interest.

What do you mean by cross-bind??? You mean the antibodies binding each other? Although there might be some background this should not happen as the antibodies should be specific for a protein, and that is what they will bind. Of course you need the appropriate controls, as well as a lot of optimisation to make sure which are the optimal concentrations of antibody to give the best signal to noise ratio.

Sorry, I dont have any references on how to do that, but I think you can maybe look for "sandwich immunoassay", or "dip stick test" (like the pregnancy one).

Finally, do you mind me asking why are you favouring this type of Dot-blot to a sandwich ELISA or even standard dot-blot where you blot the protein?

#9 Fribytter

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Posted 06 July 2011 - 11:20 AM

Thanks for your reply.

I will try to see if anybody else have been doing this.

The reason for doing this, is to make things simple. I work with some cells which I try to treat with different chemicals, and with that I have (with western blots) successfully recognized some (actually seven) different proteins which are being expressed or upregulated during the exposure with the chemical. Now the assay is set, but I would like to have a fast and easy way of measuring hundreds of chemicals effect on these seven proteins.
This means that I will need cell pellets (lysates) from every cell sample treated, but this means also that every cell lysate should be distributed into seven different Western-blot membranes for detecting the different seven protein individually.
Now my target is to have a, as you mentioned, "dip stick" which I can expose to the cell lysates, and this will give me their relative expression of my proteins of interest.

Off course sandwich ELISA could be used, (though require alot of antibodies), but I am just affraid that this will take a lot of time to do.

Hope this describes my problem...




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