Western Blot Antibody Customer Review for ARF GAP1 Polyclonal Antibody (STJ91671)


Target information

ARF GAP 1 (ADP-ribosylation factor GTPase-activating protein 1) is an enzyme that is encoded by the ARFGAP1 gene in humans. It is a GTPase-activating protein that plays a role in membrane trafficking and vesicle transport. The ARF GAP1 polyclonal antibody detects endogenous levels of ARF GAP1 protein.

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1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: ARF GAP1 Polyclonal Antibody (STJ91671)
2. What was used? Primary antibody: STJ91671 ARF GAP1 Antibody Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot Materials for validation: Mardin-Darby Bovine Kidney Cells (MDBK). (It is noted that the antibody isn¡¯t currently validated for use in bovine cells.) Click for product data sheet PDF
3. What Was The Protocol? Treatment of Materials MDBK cells were harvested at 70% confluency by trypsination, then washed (2 x 1 mL PBS), resuspended in fresh PBS (1 mL) and finally lysed by Dounce homogenisation. Gel Electrophoresis 4-20% polyacrylamide gel (pre-cast, Thermo-Fisher cat: 0025269), constant voltage 150 V, for 45 minutes. Transfer 0.45 um PVDF membrane (GE healthcare life sciences, Amersham HyBond). Semi-dry transfer, constant current 74 mA, for 60 minutes. Blocking 1X TBST with 5% skimmed milk powder, 2.5 hours (15 mL). Primary Antibody Probing Primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4?C and left overnight (2 mL) on Spiramix. Membrane Wash Membrane was washed with 1X TBST 5 times, for 10 minutes each . Secondary Antibody Probing Secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with a 1:2000 dilution ratio, for 1 hour (2ml). Membrane Wash Membrane was washed with 1X TBST 5 times, for 10 minutes each . Visualization ECL visualization ¨C BIORAD clarity ECL reagent. Blot was exposed to ECL for 2 min, then visualized using Syngene gel-doc system.
4. Gel electrophoresis information: 10% polyacrylamide gel, Semi-dry transfer, constant current 74 mA, for 60 minutes. Transfer information: 0.45 um PVDF membrane, semi-dry transfer No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualization time 1 Protein marker Geneflow BLUeye 2.5 uL n/a n/a n/a (Not validated, literature suggests 45 kDa) 2 min 2 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 3 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 4 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 5 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 6 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 7 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 8 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 9 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min 10 MDBK lysate 2.5 uL n/a 1:1000 1:2000 2 min
5. What were the results?
6. What did the customer think? Antibody Specificity: Antibody RPPA Rating: Testing results were provided independently by S. Allman at The Open University. They stated ¡°Strong target bands were observed in MDBK cells. Expected molecular mass 45 kDa matches observed band. One significant non-specific binding event was noted, but it is distinct.¡±
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