Technical Support Questionnaire – Western Blot

Name: GPCR GPR48 Antibody (2D1)Catalog #: NBP2-00974Lot Number: A01PO/Order Number: .

Species, Tissues or Cell Lines Tested: human embryonic stem cell, hESCs; RIPA extracted, supernatant; RIPA extracted, pellet (aggregate)Test Sample Preparation: 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. the samples were sonicated (or no sonication)for 15 min on ice to prevent the protein from aggregation the samples (20 ug) were heated at 37°C and 95°C in the reducing sample buffer before applying the

samples into the well. ps: positive control: 10 ug for treatment

Antibody Storage Conditions: store at -20 degreeGel/Electrophoresis Conditions: 8 % SDS-PAGE, 60 V 30 min/ 90 V, 90 minTransfer Conditions:

Please upload an image of your western blot by clicking on the center of the box.

24.7 mM Tris base, 191.8 mM glycine and 10 % methanol; 45 V, O/N, then 100 V 30 minBlocking Solution & Duration: blocking at room temperature for 1 hrblocking solution contents: 137.0 mM NaCl, 19.9 mM Tris, 5 % milk, pH 7.6

Primary Antibody Diluent and Dilutions Tested: dilute with TBST buffer (containing 0.1 % tween 20) in the ratio of 1:500Primary Antibody Incubation Time and Temperature: 2 hr at room temperature, then keep it in the cold room, O/NWash Solution Composition, Repetitions & Times: wash the membrane with TBST buffer (19.9 mM Tris, 137.0 mM NaCl, 0.1 % Tween 20, pH7.6) 3 times, 5 min for each timeSecondary Antibody Manufacturer, Host Species, Dilution, & Diluent: Jacson immunoResearch, code No.: 115-035-008-100 ul, goat anti mouse IgG.dilute with TBST (0.1 % tween 20) in the ratio of 1: 10000Secondary Antibody Incubation Time & Temperature: 1 hr at room temperatureWash Solution Composition, Repetitions, & Times: wash the membrane with TBST buffer 5 times, 5 min for each time(19.9 mM Tris, 137.0 mM NaCl, 0.1 % Tween 20, pH7.6)Detection System, Procedure & Development Time: 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 sMolecular weight of band(s): markers: thermo 26616Controls: positive control from Novus, NBP2-06791 Lot No.0812

Observations:

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).