Author: Michelle Farquhar.
Biotinylation protocol for adherent cells
Notes
a)
Sulfo-NHS-SS-biotin (Pierce, product number 21335) is an
approximately 0.24 kD molecule which reacts with amine groups, thus labels
lysine residues and the primary amine at the N-terminus of proteins. The
reagent is largely membrane impermeable, thus predominantly labels cell
surface proteins.
b)
Sulfo-NHS-SS-biotin is kept dessicated at -20 deg.C. Remove from
the freezer about one hour before use to equilibrate to room temperature –
the reason for this is that the biotin is highly moisture sensitive; as such, the
bottle should be opened for as short a time as possible during weighing.
c)
This protocol is designed to lyse cells in two different detergents: 1%
Brij97 containing Ca2+ and Mg2+ which is thought to preserve tetraspanintetraspanin interactions; and 1% Triton X-100 containing EDTA which is
thought to disrupt tetraspanin-tetraspanin interactions.
Protocol
1. Plate out the cells, at least one day before the experiment, on 6-well plates.
Allow approximately one semi-confluent well per transfection.
2. On the day before the experiment, mix the following in a tube for each
antibody (this will be enough for one Brij and one Triton i.p.):
Triton X-100 lysis buffer
750 ml
Protein G sepharose (50% slurry in PBS)
50 ml
Antibody
2.5 mg
Rotate overnight at 4 ○C to coat the antibody to the protein G sepharose.
3. On the day of the experiment, remove biotin from the freezer to warm up
(needs one hour at room temperature).
4. To biotinylate the cells, first wash the cells three times with PBS and then
add 1 ml PBS containing 1 mg/ml biotin to each well. Place on a rocker at
room temperature for 30 minutes.
5. Aspirate biotin solution and wash cells once with PBS containing 100 mM
glycine to quench the biotinylation reaction. Wash twice more with PBS.
6. Scrape off cells from each well in 1 ml PBS. Pool cells and aliquot into 1.5
ml tubes (one per i.p.).
7. Centrifuge cells, aspirate supernatant and place tubes on ice. Lyse each
cell pellet with 1 ml ice-cold Brij97 or Triton X-100 lysis buffer by vortexing for
10 seconds and then leaving on ice for 30 minutes.
8. Centrifuge on maximum speed at 4 ○C for 10 minutes to pellet nuclei and
insoluble material.
9. Remove supernatants into fresh tubes containing 20 ml protein G
sepharose (50% slurry in PBS). Vortex briefly and then rotate at 4 ○C for 30
minutes to pre-clear.
10. During the pre-clear, wash the overnight antibody-coupled beads (from
step 2) three times with 1 ml Triton X-100 lysis buffer. For each wash,
maintain the tubes on ice, vortex the tubes for 10 seconds and centrifuge at 4
○C at 5,000 rpm for 20 seconds. During the final wash, split each tube into 2 x
400 ml in fresh tubes (one labelled Brij and the other Triton). After this final
wash, place the ‘dry’ beads on ice.
11. At the end of the pre-clear, centrifuge tubes on maximum speed at 4 ○C
for 1 minute to pellet the protein G sepharose. (At this stage, a sample of
whole cell lysate should be taken). Pipette the supernatant into the tubes
containing the antibody-coupled beads. Vortex briefly and then rotate at 4 ○C
for 90 minutes.
12. Wash the beads four times with 1 ml ice-cold Brij97 or Triton X-100 lysis
buffer. For each wash, maintain the tubes on ice, vortex the tubes for 10
seconds and centrifuge at 4 ○C at 5,000 rpm for 20 seconds. During the final
wash, transfer the beads in lysis buffer to a new tube – small amounts of
protein can stick to the sides of tubes and result in non-specific bands.
13. Add 50 ml non-reducing sample buffer to each tube and store at -20
deg.C until use.
14. Boil the samples for 5 minutes before running 20 ml on a 4-12% gel – a
gradient gel is necessary to observe both large and small interacting proteins.
15. Transfer proteins to PVDF membrane and block in 3% BSA in TBST on a
rocker, either for at least one hour at room temperature or 4 deg.C overnight.
Note that milk cannot be used to block as this gives a high background with
streptavidin blotting.
16. Incubate blot with streptavidin-HRP in 3% BSA in TBST (no azide) on a
rocker for 60 minutes.
17. Wash the blot twice briefly with TBST, then five times with high salt TBST
for 6 minutes each wash. Finally wash briefly in TBS before development with
ECL/film. Note that high salt TBST is now used for washing all westerns –
this reduces background without impairing specific signals.
Buffer recipes
TBST
20 mM Tris
137 mM NaCl
0.1% Tween
pH 7.6
temp)
(lab stocks of 10x and 1x at room
3% BSA + 0.1% azide in TBST
BSA
Sodium azide
TBST
18 g
0.6 g
up to 600 ml
(store in cold room)
145 ml
up to 2 l
(store at room temp)
TBST high salt
5 M NaCl
TBST
TBS
20 mM Tris
137 mM NaCl
pH 7.6
(store at room temp)
Mike Tomlinson (August 2008)