Antibody

[arc1479]

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Now, I work on the heart, and we make primary cultures of cardiomyocytes from Rats on a regular basis. CMCs are terminally differentiated, hence no cell lines are available for any species, so I can't answer the question I am asking in any other species but the Rat (and perhaps the mouse but you don't get many cells from it).

The antibody to the protein I am interested in is only available as a human variant, with poor homology to the rat and mouse (have searched plenty). I have tried one without success. What are my options for studying perturbations, at the protein level, in Rat cells? As far as I can tell, I could either attach a FLAG tag or something to it or I could try to generate an Ab myself in the Rat. Now, I don't want to go down the route of making an Ab myself. And with the FLAG tag approach would the question of what happens to the endogenous protein come back and haunt me later (when it comes to reviewers)?

Are there any other options to consider?

 

[tr]

What are my options for studying perturbations, at the protein level, in Rat cells? As far as I can tell, I could either attach a FLAG tag or something to it or I could try to generate an Ab myself in the Rat. Now, I don't want to go down the route of making an Ab myself. And with the FLAG tag approach would the question of what happens to the endogenous protein come back and haunt me later (when it comes to reviewers)?

Just make the antibody. Your experimental options are severely limited without it, and your results will always be open to doubt. It's really not a big deal to make a peptide antibody. Just order a good-looking peptide from a peptide services co. (we use GlobalPeptide; consult a textbook or website if you don't know how to determine antigenicity, and order two different peptides to be safe) and mail it to a commercial antibody services co. (we use Covance). You'll have your first bleed in six weeks. PM me if you have questions about the process.

 

[arc1479]

Just make the antibody. Your experimental options are severely limited without it, and your results will always be open to doubt. It's really not a big deal to make a peptide antibody. Just order a good-looking peptide from a peptide services co. (we use GlobalPeptide; consult a textbook or website if you don't know how to determine antigenicity, and order two different peptides to be safe) and mail it to a commercial antibody services co. (we use Covance). You'll have your first bleed in six weeks. PM me if you have questions about the process.

How expensive is the processnfor generating an Ab de-novo? What are the chances of success?

 

[dr.z]

Just make the antibody. Your experimental options are severely limited without it, and your results will always be open to doubt. It's really not a big deal to make a peptide antibody. Just order a good-looking peptide from a peptide services co. (we use GlobalPeptide; consult a textbook or website if you don't know how to determine antigenicity, and order two different peptides to be safe) and mail it to a commercial antibody services co. (we use Covance). You'll have your first bleed in six weeks. PM me if you have questions about the process.

Its always better to have antibody against endogenous one. You can make a construct with your protein attached to FLAG tag, but it may not function like the endogenous. So I suggest getting the antibody made. There are many companies that does this. While you are waiting for the antibody to be made, since it takes time, why don't you start doing some preliminary stuff with FLAG tagged protein.

 

[SubAtomicStringTheory]

Don't try to make your Ab in a rat for a rat protein, you probably will not get a good Ab response Try using rabbit, you should get a better titer and it is a bigger animal and you should be able to get more serum..

 

[dr.z]

How expensive is the processnfor generating an Ab de-novo? What are the chances of success?

If you ask a company to do it, I have seen polyclonals from $600 and monoclonals for $4,000. Price vary by company.

Antibody may not always work for things you need since some of them use ELISA to test antibody. We had some made, and by ELISA the anitbody worked. However, they never worked for Westernblots. You have to check with company that you order from to see what their policy is.

 

[Myempire1]

What are you trying to look at? Location of the protein or just level? If you are intersted in levels, you could at least correlate mRNA levels with and mRNA in situ.