Dear all,
We are working on detecting the overexpression of Connexin43 in a Knock-in mouse model, i tried the plasma membrane fraction protocol of our lab but we couldnt detect a difference in the amout of the protein between the overexpressing mice and the wild-type mice, although there was a significant difference between the 2 with the Whole cell lysis preparation. We would like to prove that these proteins are in fact overexpressed in the membrane and not only in the cytoplasm. I will write down the protocol we used, if anyone has any comment on it or a suggestion of another protocol, please share it with us.
"1. Collect the tissue in eppendorf tubes.
2. Add 350 ul of Homogenization buffer(provided with plasma membrane isolation kit, supplemented with halt inhibitors 100 ul/10 ml)
3. Break the tissue with plastic pestles. Prepare a tissue suspension.
4. Sonicate on an ice-cooled water bath for 30 S x 3 times. Incubate on ice for 1 min between two sonications.
5. Centrifuge, 11000G, 30min. Collect the supernatants in a separate tubes (these are cytoplasmic fractions. Label them accordingly) 6. Add lysis buffer containing halt inhibitors (200 ul) (4°C) to all the pellets. Pipette up and down while adding the buffer, so that the pellet is suspended in it. 7. Sonicate again. Same procedure as before.
8. Use a chilled needle (30 G) and syringe to disrupt remaining tissue chunks (approx 5 cycles). Try to take up all the lysate each time and avoid foaming.
(Not necessary: Centrifuge 1000Gfor 2 min. Collect the supernatant (total membrane fraction))
Lysis buffer used:
Lysis buffer (modification from RIPA buffer) 50mM Tris 150mM NaCl 0,5 % NP 40 (Nonidet P-40) 0,5 % from 10 %-stock solution of NaDOC (Na-Deoxycholat) 1 % Triton x-100 Ad 500 ml, pH 7,5, stored at 4°C"
I greatly appreciate your help.
Best,
Ouss
Have you considered doing immunofluorescence microscopy or immunocytochemistry on thin sections of tissue?
Dear Adam B Shapiro ,
Yes we actually did immunofluorscence microscopy and detected the presence of more Cx43 in the membrane than in WT, we also have seen a peptide marker (P2A, which is the marker of the gene inserted Cx43 in the Cx43 overexpressing mice) presence on the membrane, which we could also only detect in the whole cell lysis and not plasma membrane fraction in Western Blot.
Do you see something wrong with the protocol?
I greatly appreciate your help.
Yours,
Ouss
I was thinking that if the expression level wasn't high enough to see against the background in a Western blot, due to the limited amount of protein that can be added in a gel lane, it might show up better by the other methods.
We have detected the desired protein Cx43 in both WT-mice and Cx43-overexpressing mice, the problem was that there was no difference in the amount of the protein detected in WB between the 2 mice, although it should be more for the Cx43-overexpressing mice.... We are looking for a solution.
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