Lot# 1934474

TECHNICAL
DATA
SHEET
Membrane Target Systems™
Caution: For Laboratory Use. A research reagent for research purposes only
human Serotonin 5-HT2B Receptor
Product No.: ES-314-M400UA
Lot No.: 1934474
Material Provided
Membranes:
1 x 400 units / 400 µL frozen aliquot
Product Information
Cellular Background:
CHO-K1
GenBank Accession Number:
X77307
Unit Size:
7 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):
1.13
Expression Level (BMAX):
3.8 pmol/mg membrane protein.
3
KD for [ H]-LSD :
0.41 nM
Protein Concentration:
7 µg/µL
(1)
Smith, P.K., et al. (1985). Anal. Biochem. 150, 76-85.
TDS-ES-314-M400UA-04
Page 1 of 4
Recommended Assay Conditions
Assay Buffer:
50 mM Tris pH 7.4, 4 mM CaCl2, 0.1% ascorbic acid
Wash Buffer:
50 mM Tris-HCl pH 7.4
Binding Protocol:
Binding assays are performed in 550 µL total volume according
to the following conditions:
1 - Membrane dilution: 0.05 mL of membranes + 24.95 mL assay buffer (1:500 dilution)
2 - Incubation:
25 µL of incubation buffer or Serotonin (5-HT) (Sigma H9523) 50 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:
30 minutes at 37 °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
4000
T o ta l
N o n s p e c ific
B o u n d (c p m )
3000
S p e c ific
2000
1000
0
0
1
2
3
4
5
6
7
8
[3 H ]-L S D (n M )
Figure 1: Saturation binding assay curve (filtration)
96-well saturation binding assay curve (7 µg
3
membranes/well,
TopCount®)
using
[ H]-LSD
(PerkinElmer NET638 Lot No.: 1930614)
TDS-ES-314-M400UA-04
Page 2 of 4
Typical Product Data
Bound (cpm)
2000
Spiperone
1500
Yohimbine
Ritanserin
1000
BW 723C86
500
0
-
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
[Compound] (M)
Figure 2: Competition binding assay curve (filtration)
96-well competition binding assay curve (7 µg
membranes/well,
TopCount®).
Recommended
radioligand concentration = 1 nM.
*Even though two sites can be observed occasionally
with some ligands, the data presented is derived from
single site fitting.
Reference Compounds
Spiperone
TDS-ES-314-M400UA-04
Page 3 of 4
Ki
(nM)
813
Yohimbine
29
Ritanserin
0.60
BW 723C86
47
Suggested Materials and Instrumentation
Please visit our website
www.perkinelmer.com/GPCR
This product is not for resale or distribution except by authorized distributors.
When applicable, the CMV promoter is covered by U.S. Patents 5,168,062 and 5,385,839, licensed to
PerkinElmer by the University of Iowa research Foundation
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