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