Biology Reference
In-Depth Information
chloroform by putting the container on a vacuum system overnight. Determine
the amount of lipid by weighing the flask and subtraction of the empty weight.
￿ Add 4.8 mL 10% DM to the flask with 57.6 mg dried DOPS (1.2% final
concentration) and disperse the lipid using a water bath sonicator. The resulting
opalescent solution is cleared by one or two freeze-thaw cycles. The clear
solution is filtered with a 0.2
m syringe filter using a glass syringe (or methanol
washed disposable plastic syringe) and stored at
m
20 C.
￿ Preparation of buffer N. Mix 850
L DOPC (1.06% in 10% DM), 330
L
m
m
DOPS (1.2% in 10% DM), 460
L CHS
(4.65% in 19.7% CHAPS), 6.43 mL 78% glycerol, 2.5 mL 2 M (NH 4 ) 2 SO 4 ,
and 1 mL 1 M Tris-HCl buffer, pH 7.0. Add purified water to 50 mL final
volume. Filter with 0.2
L 10% DM, 80
L 20% CHAPS, 750
m
m
m
20 C. Use this
buffer for solubilization and purification of GPCRs, such as C-C chemokine
receptor 5 (CCR5) ( Knepp, Grunbeck, Banerjee, Sakmar, & Huber , 2011 ).
￿ Prepare buffer N2 by substituting Tris buffer with 50 mM HEPES-NaOH buffer
pH 7.5 and supplement with 2 mg/mL BSA.
m Steriflip and store aliquots at
m
15.7 PROTOCOL 5: SINGLE-MOLECULE
IMMUNOPRECIPITATION ON GLASS-BOTTOM MICROPLATES
15.7.1 Step 1: cleaning of glass-bottom microtiter plate
15.7.1.1 Materials
384-well, PS SensoPlate, Small Volume, LoBase, Glass Bottom (Greiner 788896)
384-well microplate
Dimethyl sulfoxide (DMSO, ACS grade, Fisher)
Ethanol USP 95%
Hydrochloric acid (37% HCl, ACS grade, Fisher)
Purified and filtered water (Milli Q Synthesis)
Plastic bottle with sprayer
Filtration unit (e.g., Millipore Steritop-GP)
Ultrasonic cleaning bath (Bransonic)
Automated pipetting system (epMotion 5070, Eppendorf) with 50
L
m
multichannel tool (TM-50) or manual multichannel pipette (1-20
L, Pipetman)
m
Benchtop centrifuge with microplate swinging bucket rotor
Whatman filter paper
Polypropylene ziplock plastic bag (11
16 cm)
15.7.1.2 Solutions and buffers
70% FILTERED ETHANOL:
365 mL 95% ethanol.
Add purified water to 500 mL.
Search WWH ::




Custom Search