human Muscarinic M3 Receptor

TECHNICAL
DATA
SHEET
Membrane Target Systems™
Caution: For Laboratory Use. A research reagent for research purposes only
human Muscarinic M3 Receptor
Product No.: RBHM3M400UA
Lot No.: XXX-XXX-X
Material Provided
Membranes:
1 x 400 units / 1000 µL frozen aliquot
Product Information
Cellular Background:
CHO-K1
GenBank Accession Number:
NM_000740
Unit Size:
15 µg protein / unit
Storage Buffer:
50 mM Tris-HCL (pH 7.4), 0.5mM EDTA, 10mM MgCl2, 10%
sucrose.
Storage Conditions:
Store at -80°C. Freeze-thaw is not recommended as it can
affect product performance and homogeneity. In order to
minimize negative impact of freeze-thawing, flash freeze in liquid
o
nitrogen for 30 seconds prior to transferring to -80 C.
Stability:
This product is stable for at least 3 years from reception if used
and stored under recommended conditions.
Quality Control
Bmax and Kd are determined using radioactive saturation binding assays (Figure 1). Protein
(1)
concentration is determined using the BCA method . Ratio-to-Reference (RTR) is determined by
dividing the maximal signal of the current lot (Bmax in fmoles) by the maximal signal of a pre-defined
reference tested in parallel. RTR is an indicator of lot-to-lot consistency. *We certify that these results
meet our quality release criteria.
Ratio-to-Reference (RTR):
N/A
Expression Level (BMAX):
11.98 pmol/mg membrane protein.
3
KD for [ H]-Scopolamine (N-methyl) : 0.15 nM
Protein Concentration:
(1)
6 µg/µL
Smith, P.K., et al. (1985). Anal. Biochem. 150, 76-85.
TDS-RBHM3M400UA-04
Page 1 of 4
Recommended Assay Conditions
Assay Buffer:
PBS pH 7.4
Wash Buffer:
50 mM Tris-HCl pH 7.4, 154 mM NaCl
Binding Protocol:
Binding assays are performed in 550 µL total volume according
to the following conditions:
1 - Membrane dilution: 0.125 mL of membranes + 24.875 mL assay buffer (1:200 dilution)
2 - Incubation:
25 µL of incubation buffer or Atropine (Sigma A025) 5 µM final for non
specific binding (Saturation binding assay)
For competition binding assay: 25 µL of reference compounds at
decreasing concentrations (see figure 2)
25 µL of radioligand at the appropriate concentration (see graph below)
500 µL of diluted membranes
3 - Incubation time:
120 minutes at 27 °C
4 - Filtration:
aspirate and wash 9 x 500 µL with ice cold wash buffer over GF/C filter
(presoaked in 0.5 % PEI).
Lot Specific Data
30000
T o ta l
B o u n d (c p m )
N o n s p e c ific
S p e c ific
20000
10000
0
0
1
2
3
4
5
6
7
8
9
10
[ 3 H ] - S c o p o la m in e ( N - m e t h y l) ( n M )
Figure 1: Saturation binding assay curve (filtration)
96-well saturation binding assay curve (15 µg
3
membranes/well, TopCount®) using [ H]-Scopolamine
(N-methyl) (PerkinElmer NET636 Lot No.: 1885805)
TDS-RBHM3M400UA-04
Page 2 of 4
Typical Product Data
Bound (cpm)
5000
4000
p-Fluorohexahydro-sila-difenidol
3000
Pirenzepine
2000
Methoctramine
1000
0
-
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
[Compound] (M)
Figure 2: Competition binding assay curve (filtration)
96-well competition binding assay curve (15 µg
membranes/well,
TopCount®).
Recommended
radioligand concentration = 0.6 nM.
*Even though two sites can be observed occasionally
with some ligands, the data presented is derived from
single site fitting.
Reference Compounds
p-Fluorohexahydro-sila-difenidol
TDS-RBHM3M400UA-04
Page 3 of 4
Ki
(nM)
87
Pirenzepine
637
Methoctramine
1997
Suggested Materials and Instrumentation
Please visit our website
www.perkinelmer.com/GPCR
This product is not for resale or distribution except by authorized distributors.
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