Date post: | 13-Apr-2017 |
Category: |
Science |
Upload: | st-johns-laboratory-ltd |
View: | 87 times |
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What did the customer think?Antibody Specificity:
Antibody RPPA Rating:
Testing results were provided independently by J. Huang at Newcastle University. They stated “HeLa cell lysates were used. Western blot signals were detected using an Odyssey (Li-Cor) machine). Four positive protein bands were detected around the size of assumed Bim protein from 17 to 25KDas using anti-Bim (STJ91855) antibody at 1:500 dilution and a rabbit Alexa fluor 680 secondary antibody (in red).”
(KD)
46
32
57
76
100
134
2225
Bim
What was used?Primary antibody: STJ91225 Anti-Bim antibody
Provider: St John's Laboratory Ltd.
Dilution ratio: 1:500
Application: Western Blot
Materials for validation: Lysates from Hela cell line
Click for product data sheet PDF
What Was The Protocol?Treatment of Materials When cells were cultured to 70%-80% plating density, lysis buffer with protease inhibitors
(Sigma) and equal volume of 2X loading buffer were added and heated at 100˚C for 10 minutes. Supernatant was collected after centrifugation at 12,000 rpm for 25 minutes at 4°C and used for 10% Bis-Tris plus gel for protein separation.
Gel Electrophoresis 10% polyacrylamide gel, constant voltage 160 V, 60 minutes.
Transfer 0.45 µm PVDF membrane, 1 piece of gel (wet transfer), constant current 80 Amp for 90 minutes.
Blocking 1X Odyssey blocking solution for 2 hours at RT
Primary Antibody Probing Primary antibody was added to the 1X Odyssey blocking solution with 1:500 dilution, and the membrane was incubated at 4˚C overnight with agitation.
Membrane Wash 1X TBST wash 3 times
Secondary Antibody Probing Secondary antibody was added to 1X Odyssey blocking solution with 1:1000 dilution ratio, for 1.5 hours at RT
Membrane Wash 1X TBST wash 3 times
Visualization Fluorescent signals were visualized using a Li-Cor Odyseey machine using 700nm and 800nm detecting channels
What was the protocol?10% polyacrylamide gel, constant voltage 160 V, 60 minutes.
0.45 µm PVDF membrane, 1 piece of gel (wet transfer), constant current 80 Amp for 90 minutes.
No. Antigen Loading amount
Primary antibody
Primary antibody
dilution ratio
Secondaryantibody
dilution ratioTarget band KD Visualization
time
1 Hela lysate 10µg (total protein)
STJ91855 anti-Bim Antibody
1:500 1:0000 ~22/25KD N/A
2 ~46KD N/A1:0000Actin-beta 1:1000
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