I'd start by cloning the region into an expression vector in E. coli and doing the basic biochemistry. Talk with your research mentor. Good luck!

Dear Lisa,

several things deserve attention and need to be discussed / clarified:

1. you mentioned that your lymphoblastoid cell line is heterozygous, so this means if your hypothesis is correct it makes a mixture monomers and dimers.

2. Do you have a control, i.e. a homozygous lymphoblastoid cell line?

3. What is known in the literature about the stability of the homodimer?

4. Has the structure been solved, i.e. can you look at it and e.g. highlight the part that is missing in your variant to support your hypothesis?

5. Do you have specific antibodies for sensitive detection?

6. Is the protein available commercially as recombinant protein, e.g. to use as a control?

7. Is the protein secreted or intracellular?

8. Can you reveal more details about your protein? Remember, the more specific your question the more specific the answers you'll be getting.

You may be able to test your hypothesis by running a size exclusion chromatography, collect the fraction and test them for presence of your target protein. By comparison with the control (native target protein) and the size markers (i.e. calibrated SEC) you can deduce the oligomeric state.

Good luck!

Dear Markus Sack ,

Below my answers. Thank you very much for your kind help!

1. you mentioned that your lymphoblastoid cell line is heterozygous, so this means if your hypothesis is correct it makes a mixture monomers and dimers.

I imagine 4 possibilities:

- dimers of WT protein

- dimers of WT-mutant protein --> this could cause a dominant negative effect ?

- dimers of mutant-mutant protein

- monomers of mutant or WT protein

2. Do you have a control, i.e. a homozygous lymphoblastoid cell line?

Yes.

3. What is known in the literature about the stability of the homodimer?

There's only a work, in which the authors showed that it is a stable complex able to interact with other proteins ( 2 molecules of protein X and 2 molecules of protein Y independently or at the same time)

4. Has the structure been solved, i.e. can you look at it and e.g. highlight the part that is missing in your variant to support your hypothesis?

The structure has been partially solved, but this allowed me to design the mutant and WT protein on Phyre2.

5. Do you have specific antibodies for sensitive detection?

Do you mean an antibody able to discriminate between the two forms? No, I have only an antibody that should recognize both the forms.

6. Is the protein available commercially as recombinant protein, e.g. to use as a control?

No

7. Is the protein secreted or intracellular?

It's a cytoplasmic protein

8. Can you reveal more details about your protein? Remember, the more specific your question the more specific the answers you'll be getting.

I hope that the info I gave you will be sufficient :)

Dear Katie A Burnette

Thank you for your answer. I would like to use patient-derived cell, since the variant is in heterozygosis and I imagine a particular mechanism (see the answer to Markus) that I would lose if I used a construct. Anywat, I could use two contructs and co-transfect them with 2 different tags.