HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: ARF GAP1 Polyclonal Antibody (STJ91671)

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

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 µm 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 – BIORAD clarity ECL reagent. Blot was exposed to ECL for 2 min, then visualized using Syngene gel-doc system.

Gel electrophoresis information: 10% polyacrylamide gel, Semi-dry transfer, constant current 74 mA, for 60 minutes.Transfer information: 0.45 µm PVDF membrane, semi-dry transfer

No. Antigen Loading amount Primary antibody

Primary antibody

dilution ratio

Secondaryantibody dilution

ratioTarget band KD Visualization time

1Protein 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

What were the results?

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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