1. Technical Support Questionnaire – Western Blot
Name:Click here to enter text.
GPCR GPR48 Antibody (2D1)
Catalog #:Click here to enter text.
NBP2-00974
Lot Number:Click here to enter text.
A01
PO/Order Number:Click here to enter text..
Please upload an image of
your western blot by clicking
on the center of the box.
Species, Tissues or Cell Lines Tested:Click here to enter text.
human embryonic stem cell, hESCs; knock down, shLGR4, negative control
Test Sample Preparation:Click here to enter text.
the cell pellet was lysed by RIPA buffer, then Keep it in the cold room for 1.5 hr with 10 rpm rotation, followed
by centrifugation at 13000 rpm for 20 min, transfer the supernatant into a new centrifuge tube
Antibody Storage Conditions:Click here to enter text.
store at -20 degree
Gel/Electrophoresis Conditions:Click here to enter text.
8 % SDS-PAGE, 60 V 30 min/ 90 V, 90 min
Transfer Conditions:Click here to enter text.
24.7 mMTris base, 191.8 mM glycine and 10 % methanol; 40 V, O/N, then 100 V 30 min
Blocking Solution & Duration:Click here to enter text.
blocking at room temperature for 1 hr
blocking solution contents: 137.0 mMNaCl, 19.9 mMTris, 5 % milk, pH 7.6

2. Primary Antibody Diluent and Dilutions Tested:Click here to enter text.
dilute with TBST buffer (containing 0.1 % tween 20) in the ratio of 1:500
Primary Antibody Incubation Time and Temperature:Click here to enter text.
2hr at room temperature, then keep it in the cold room, O/N
Wash Solution Composition, Repetitions & Times:Click here to enter text.
wash the membrane with TBST buffer (19.9 mMTris, 137.0 mMNaCl, 0.1 % Tween 20, pH7.6) 3 times, 5 min for
each time
Secondary Antibody Manufacturer, Host Species, Dilution, & Diluent:Click here to enter text.
JacsonimmunoResearch, code No.: 115-035-008-100 ul, goat anti mouse IgG.
dilute with TBST (0.1 % tween 20) in the ratio of 1: 10000
Secondary Antibody Incubation Time & Temperature:Click here to enter text.
1 hr at room temperature
Wash Solution Composition, Repetitions, & Times:Click here to enter text.
wash the membrane with TBST buffer 5 times, 5 min for each time
(19.9 mMTris, 137.0 mMNaCl, 0.1 % Tween 20, pH7.6)
Detection System, Procedure & Development Time:Click here to enter text.
add 1000 ul of ECL substrate (thermo, femto) onto the membrane. then cover the membrane with a
transparent plastic membrane, followed by luminescence detection by FUJI FILM LAS-4000 with a exposure
time of 30 s
Molecular weight of band(s):Click here to enter text.
markers: thermo 26616
Controls:Click here to enter text.
positive control from Novus, NBP2-06791 Lot No.0812
Observations:Click here to enter text.
In human embryonic stem cell and positive control provided by your company (Novus), we can not see the
signal from the predicted size (about 100 kDa).