Biology Reference
In-Depth Information
3.
Detection of 5-HT
4
R dimers by coimmunoprecipitation
4.
Analysis of 5-HT
4
R dimers by immunofluorescence
5.
Analysis of 5-HT
4
R dimers by time-resolved F¨rster resonance energy transfer
(TR-FRET)
7.1
MATERIALS
1.
PBS: phosphate-buffered saline, pH 7.4, LONZA, DPBS,10
without Ca
2
þ
and Mg
2
þ
, #BE17-515F.
2.
Trypsine-EDTA solution 1
, Life Technologies, #2530096.
: electroporation mix composed of 50 mM K
2
HPO
4
,20mMCH
3
CO
2
K,
20 mM KOH, and 26.7 mM MgSO
4
, in water. Adjust the pH to 7.4 with
acetic acid.
4.
0.4 cm electroporation cuvettes, Eurogentec, #CE-0004-50.
5.
Falcon 100 or 150 mm cell culture dishes, cluster of 12, 24, or 96 wells, 12 mL
Falcon tubes (BD Biosciences).
6.
Microtubes: 1.5 mL capacity.
7.
DMEM-10% DFBS: DMEM (DMEM 4.5 g/L glucose with
L
-glutamine,
LONZA, #BE12-604F) supplemented with 10% dialyzed fetal bovine serum
(DFBS) (Lonza, #14-810F).
8.
Myc-tagged and HA-tagged 5-HT
4
receptors cDNA in plasmid suitable for
expression in mammalian cells (e.g., pRK5 or pcDNA3.1). Epitopes are
located at the N-terminus of the receptor. The signal peptide (SP) from the
metabotropic glutamate receptor type 5 is added before HA tag whereas no SP
is necessary with Myc-tag.
9.
Empty plasmid cDNA used as carrier and control (e.g., pRK5 or pcDNA3.1).
10.
Rubber policeman: 34 mm wide rubber scrapper “Model K” on Saint-Gobain,
Verneret Plastic catalog, #V101029, ROGO-SAMPAIC (France), or
AUXILAB S.L (Spain).
11.
Potter homogenizer: 1 mL capacity PYREX
®
Potter tissue homogenizer,
Corning, #7725T-1.
12.
Protease inhibitors: cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets,
Roche Applied Science, #04693132001.
13.
Tris Lysis Buffer: 10 mM Tris-HCl, pH
3.
EP1
¼
7.4; 2 mM EDTA; protease
inhibitors.
14.
Bradford reagent: Quick Start Bradford 1
Dye Reagent, Bio-Rad, #500-0205.
15.
Solubilization buffer: 50 mM NaHPO
4
/NaH
2
PO
4
,pH
¼
7.2; 1 mM EDTA; 1%
SDS or 10 mM CHAPS; protease inhibitors.
16.
Deglycosylation buffer: 50 mM NaHPO
4
/NaH
2
PO
4
,pH
¼
7.2; 10 mM EDTA;
1% SDS or 10 mM CHAPS; protease inhibitors.
17.
N-glycosidase F: PNGase F, New England Biolabs, #P0704S.
18.
Laemmli buffer 4
: 200 mM Tris/HCl, pH
¼
6.8; 8% SDS; 40% glycerol; 20%
b
-mercaptoethanol; bromophenol blue.
