I have been attempting to do surface biotinylation on mouse tissue sections for over a year now. I run westerns on intracellular, surface and total samples. I am using NaKaATPase as the surface marker and either GAPDH and/or tubulin as surface marker.
1) I consistently identify intracellular protein in all samples, including surface sample
2) Most times I identify surface protein in all samples, including intracellular sample
I could imagine NaKaATPase may be seen in samples that have not been fully biotinylated?, but why am I always getting an intracellular band (GAPDH and/or tubulin) on my surface sample? These are true bands. I must admit that I likely load more protein on gels that some people do (for total it is 20ul of 2mg/ml). Thus my bands are strong in comparison to many published findings. I suppose if I loaded less, the band would be “hidden,” but obviously want to troubleshoot this! I have general protocol below. I have talk to Pierce and other that have done this procedure, but no advice has helped….YET. Anyone see obvious problem?
1) Brain slice preparation
a) Extract brain quickly from mouse and place in ice cold ACSF (5 min).
Cut brain slices (400um) with vibratome in ice cold gasified ACSF:
c) Wash slices two times for 5 minutes in ice cold ACSF
2) Slice Recovery: ACSF continuously gasified with 95%-O2 and 5%-CO2 (60 min at 32°C.
3) Slice biotinylation: Transfer slice into well of continuously gassed ACSF + NHS-SS biotin solution (1mg NHS-SS biotin/ml ACSF) on ice for 30 min
4) Rinse in cold quenching solution (100mg/mL glycine in PBS) two times for 5 minutes then wash 3-5 times for 5 minutes with cold ACSF and collected in a 1.5 ml tube
5) Add 20ul of tissue lysis buffer + 180ul of Buffer A (with proteinase inhibitor) into collection (total of 200ul) and sonicate to homogenize
6) Equate protein concentration levels among samples by adding Buffer A with proteinase inhibitor (2mg/ml). Set aside some of sample in separate 1.5ml tube labeled as “total” protein sample.
7) After correction for protein levels, add 40ul of Streptavidin beads (Pierce) to 150ul of sample and rotate over night at 4 °C.
8) Vortex beads then precipitate beads by centrifugation (1min, med speed -4000 rpm).
9) Separate supernatant from beads and save in separate 1.5ml tubes labeled as intracellular protein sample.
a) Wash beads three times with cold Buffer A and let for 5 minutes inverting throughout. Centrifuge at 4C for 5 minutes
wash beads twice with Washing Buffer and for 5 minutes inverting throughout. Centrifuge at 4C for 5 minutes
c) wash beads twice with Buffer A for 5 minutes inverting throughout. Centrifuge at 4C for 5 minutes: discard supernatant and label beads as surface protein sample.
10) Proceed with westerns on total intracellular and surface samples: to identify and quantify target, surface, and intracellular bands
Among other things, we have
1) Added various washing steps
2) Added more Streptavidin beads
3) Tried different avidin beads
4) Increase biotinylation time