st john's laboratory ltd booklet 2015
TRANSCRIPT
INDEPENDENT ANTIBODY VALIDATIONREVIEWS
Improving in quality and reproducibility through antibody validation.
- St John’s Laboratory Team
St John's Laboratory was awarded "Most
Impressive Startup" by a panel of judges from
F1000 Research. St John's Laboratory was
recognized for their antibody validation project.
At St John’s Laboratory we would like to increase the transparency of our antibodies' quality, while also providing sufficient background data on the product’s performance in real-experimental conditions. Through our Premier Partner Scheme (PPS) we have been able to connect our customers with not only a product, but also the opportunity to evaluate the product’s performance before making a purchase.
Our customer’s validation review provide other end-users valuable information on:
- New applications- Optimal dilution conditions- Protocols- Images of our products at work
VALIDATION REPORT: Cdk2 Polyclonal Antibody (STJ92197)
BACKGROUND
Antibody: Cdk2 Polyclonal AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ92197Supplier: St John’s Laboratory Experimental Notes: In Hela and Jurkat cell line, there were target bands detected by STJ92197. Cdk2 Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: Actin-Beta Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
PROTOCOL
Gel electrophoresis: 10% polyacrylamide gel, constant voltage 160 V, 60 min.Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.Blocking: 1X TBST with 5% skimmed milk powder for 2 hr.Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4˚C for overnight.Membrane wash: 1X TBST wash for 3 times.Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1：10000 dilution ratio, for 1.5 hr.Membrane wash: 1X TBST wash for 3 times.Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualiza-tion time was identical for the same antigen).
Cdk2 Polyclonal Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug St J92197. Cdk2Polyclonal Antibody
1:10001:10000 32KD 60S
2 Jurkat lysate 10 ug 1:10000 32KD 60S
Figure 1: Lane1: Hela; Lane 2: Jurkat
VALIDATION REPORT: GSK3β Polyclonal Antibody (STJ93447)
BACKGROUND
Antibody: GSK3β Polyclonal AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ93447Supplier: St John’s Laboratory Experimental Notes: In Hela cell line, there was target band detected by STJ93447. GSK3β Polyclonal Antibody. In A549 cell line, there was no target band detected by STJ93447. GSK3β Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: Actin-Beta Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
Table: Validation Results for GSK3β Polyclonal Antibody
GSK3β Polyclonal Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug St J93447. GSK3β Polyclonal Antibody
1:10001:10000 47KD 30S
2 A549 lysate 10 ug 1:10000 47KD 30S
Figure 1: Lane1: Hela; Lane 2: A549
VALIDATION REPORT: mTOR Polyclonal Antibody (STJ94280)
BACKGROUND
Antibody: mTOR Polyclonal AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ94280Supplier: St John’s Laboratory Experimental Notes: In Hela and Jurkat cell line, there were target bands detected by STJ94280. mTOR Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: mTOR Polyclonal Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
Table: Validation Results for mTOR Polyclonal Antibody
mTOR Polyclonal Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug STJ94280. mTOR Polyclonal Antibody
1:10001:10000 288KD 35S
2 Jurkat lysate 10 ug 1:10000 288KD 35S
Figure 1: Lane1: Hela; Lane 2: Jurkat
VALIDATION REPORT: NFκB-p65 Polyclonal Antibody (STJ94468)
BACKGROUND
Antibody: NFκB-p65 Polyclonal AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ94468Supplier: St John’s Laboratory Experimental Notes: In Hela cell line, there was a target band detected by STJ94468. NFκB-p65 Polyclonal Antibody. In A549 cell line, there were no target bands detected by STJ94468. NFκB-p65 Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: NFκB-p65 Polyclonal Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
Table: Validation Results for NFκB-p65 Polyclonal Antibody
NFκB-p65 Polyclonal Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug STJ94468. NFκB-p65 Polyclonal Antibody
1:10001:10000 65KD 30S
2 A549 lysate 10 ug 1:10000 65KD 30S
Figure 1: Lane1: Hela; Lane 2: Jurkat
VALIDATION REPORT: p53 Polyclonal Antibody (STJ94890)
BACKGROUND
Antibody: p53 Polyclonal AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ94890Supplier: St John’s Laboratory Experimental Notes: In Jurkat cell line, there was a target band detected by STJ94890. p53 Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: p53 Polyclonal Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
Table: Validation Results for p53 Polyclonal Antibody
p53 Polyclonal Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug STJ94890. p53Polyclonal Antibody
1:10001:10000 53KD 30S
2 Jurkat lysate 10 ug 1:10000 53KD 30S
Figure 1: Lane1: Hela; Lane 2: Jurkat
VALIDATION REPORT: PCNA Polyclonal Antibody (STJ94982)
BACKGROUND
Antibody: PCNA Polyclonal AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ94982Supplier: St John’s Laboratory Experimental Notes: In Hela and Jurkat cell line, there were target bands detected by STJ94982. PCNA Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: PCNA Polyclonal Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
Table: Validation Results for p53 Polyclonal Antibody
PCNA Polyclonal Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug St J94982. PCNA Polyclonal Antibody
1:10001:10000 36KD 30S
2 Jurkat lysate 10 ug 1:10000 36KD 30S
Figure 1: Lane1: Hela; Lane 2: Jurkat
VALIDATION REPORT: GAPDH Polyclonal Antibody (STJ96417)
BACKGROUND
Antibody: GAPDH Polyclonal AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ96417Supplier: St John’s Laboratory Experimental Notes: In Hela and Jurkat cell line, there were target bands detected by STJ96417. GAPDH Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: GAPDH Polyclonal Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
Table: Validation Results for GAPDH Polyclonal Antibody
GAPDH Polyclonal Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug STJ96417. GAPDH Polyclonal Antibody
1:10001:10000 36KD 130S
2 Jurkat lysate 10 ug 1:10000 36KD 130S
Figure 1: Lane1: Hela; Lane 2: Jurkat
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VALIDATION REPORT: Actin-Beta Antibody (STJ91464)
BACKGROUND
Antibody: Actin-Beta AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ91464Supplier: St John’s Laboratory Experimental Notes: In Hela and Jurkat cell line, there were target bands detected by STJ91464.Actin-beta antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: Actin-Beta Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
PROTOCOL
Gel electrophoresis: 10% polyacrylamide gel, constant voltage 160 V, 60 min.Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.Blocking: 1X TBST with 5% skimmed milk powder for 2 hr.Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4˚C for overnight.Membrane wash: 1X TBST wash for 3 times.Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1：10000 dilution ratio, for 1.5 hr.Membrane wash: 1X TBST wash for 3 times.Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualiza-tion time was identical for the same antigen).
Actin-Beta Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug STJ91464.Actin-beta antibody
1:10001:10000 ~50KD 20S
2 Jurkat lysate 10 ug 1:10000 ~50KD 20S
Figure 1: Lane1: Hela; Lane 2: Jurkat
VALIDATION REPORT: Cdc2 Polyclonal Antibody (STJ92154)
BACKGROUND
Antibody: Cdc2 AntibodyMethod of Validation: Western Blot Validator: BPISupplier Catalog Number: STJ92154Supplier: St John’s Laboratory Experimental Notes: In Hela and Jurkat cell line, there were target bands detected by STJ92154. Cdc2 Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: Cdc2 Antibody (Dilution 1:1000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
PROTOCOL
Gel electrophoresis: 10% polyacrylamide gel, constant voltage 160 V, 60 min.Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.Blocking: 1X TBST with 5% skimmed milk powder for 2 hr.Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4˚C for overnight.Membrane wash: 1X TBST wash for 3 times.Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1：10000 dilution ratio, for 1.5 hr.Membrane wash: 1X TBST wash for 3 times.Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualiza-tion time was identical for the same antigen).
Cdc2 Antibody has been independently validated for western blot by BPI.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug St J92154. Cdc2Polyclonal Antibody
1:10001:10000 34KD 13S
2 Jurkat lysate 10 ug 1:10000 34KD 13S
Figure 1: Lane1: Hela; Lane 2: Jurkat
Figure 1: Western Blot for EGFR. Grey arrowhead indicates the expected molecular weight of ~170 kDa.PROTOCOL
Cell/tissue total protein lysates were boiled in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol at 95°C for 5 minutes prior to loading. 15 μg of boiled lysate were loaded and resolved on a 12% SDS-polyacrylamide gel. The Precision Plus Protein™ All Blue Prestained Standards from BioRad (161-0373) were used as molecular mass markers. Proteins were transferred onto nitrocellulose membrane by tank transfer and protein transfer was confirmed with Ponceau S staining. The immunoblot membrane was blocked in 2.5% skim milk and 1.5% BSA solution in TTBS at room temperature for 60 minutes. The membrane was washed in TTBS twice for 5 minutes each. The membrane was immersed with the protein side up in the antibody solution in TBS and incubated overnight at 4°C with gentle agitation. The membrane was rinsed twice with TTBS. The membrane was washed in TTBS twice for 5 minutes each. The membrane was washed in TTBS once for 15 minutes. The membrane was incubated in the HRP-conjugated secondary antibody solution in TBS for 60 minutes at room temperature with gentle agitation. The membrane was rinsed twice with TTBS. The membrane was washed in TTBS twice for 5 minutes each. The membrane was washed in TTBS once for 15 minutes. Signals were detected by chemiluminescence (ECL). The blot was scanned for 320 seconds. The membrane was rinsed three times with TTBS. Repeated Steps 4-15 with the loading control antibody and its matching secondary antibody.
VALIDATION REPORT: Epidermal Growth Factor Receptor (EGFR) antibody (STJ40568)
BACKGROUND
Antibody: Epidermal Growth Factor Receptor (EGFR) antibodyMethod of Validation: Western Blot Validator: Kinexus Bioinformatics CorporationSupplier Catalog Number: STJ40568Supplier: St John’s Laboratory Experimental Notes: A strong band was observed in the positive control at the expected size (~170 kDa) that is not observed in the negative control.
INDEPENDENT VALIDATION METHODS
Primary Antibody: Epidermal Growth Factor Receptor (EGFR) antibody (Dilution 1:1,000)Loading Control Antibody: Beta-actin; supplier: Santa Cruz (Dilution 1:200)Secondary Antibody: Sheep anti-mouse IgG-HRP; supplier: GE Healthcare (Dilution 1:10,000)
Epidermal Growth Factor Receptor (EGFR) antibody has been independently validated for western blot by Kinexus Bioinformatics Corporation under the Science Exchange network.
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VALIDATION REPORT: FAAH Antibody (STJ23602)
BACKGROUND
Antibody: FAAH AntibodyMethod of Validation: ImmunofluorescenceValidator: Natalia Malek (PhD Student)Supplier Catalog Number: STJ23602Supplier: St John’s Laboratory Experimental Notes: We completed evaluation of FAAH antibody. It worked really well, so we decided to purchase a vial from St John’s Laboratory.
INDEPENDENT VALIDATION METHODS
Primary Antibody: FAAH antibody (Dilution 1:50)
PROTOCOL
Samples were frozen and stored at -80°C until sections were cut at 12 μm thick and collected onto gelatin-coated slides (Menzel GmbH, Braunschweig, Germany). For immunofluorescence labeling, serial sections of DRG were hydrated in PB buffer (pH=7.4) for 15 mins. Cell membranes were permeabilised in PB containing 0.1% Triton X-100 and afterwards incubated for 1 h in 10% normal goat serum (NGS, Jackson ImmunoResearch Laboratories, West Grove, USA) in PB containing 0.4% Triton X-100 (blocking solution). Sections were incubated for 48 hrs at 4°C in a humid chamber with the respective primary antibodies (FAAH, St. John’s laboratory; 1:50). After three washes in PB, triple immunofluorescence was revealed by incubation at 4°C in for 24 hrs in the dark with the appropriate fluorochrome-conjugated secondary antibody. Sections were washed 3 times for 15 mins with PB and coverslipped with Aquatex mounting medium (Merck, Darmstadt, Germany). The sections processed for immunofluorescence were studied with a fluorescence microscope (DM RXA2) with confocal scanner (TCS SL) (Leica Microsystems GmbH, Manheim, Germany) equipped with the following lasers: Ar 488, He-Ne 543, He-Ne 633.
FAAH antibody has been independently validated for immunofluorescence by PhD student from Institute of Pharmacology Polish Academy of Sciences.
Figure 1: Immunofluorescence. Dorsal root ganglia stained with FAAH antibody.
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PROTOCOL
Plating the cells: 200k of HEK293 cells were plated in each wells in a 24 well plate and cultured overnight in serum free media in an incubator at 37C with 5% CO2.
Transfection Cells were transfected with pLV-mCherry using Lipofectamine 2000 according to manufacturer's protocol and grown overnight before fixing.
Fixing/permeablization/staining Cells were fixed with 4% PFA for 15 min at RT. Cells were permeablized with 0.1% Triton-X100 for 30 min. Afterwards, cells were blocked using 1% BSA for 30 min. Cells were incubated with mCherry antibody (1:200, 1 mg/mL) or isotype control (1:400, 2 mg/mL) at RT for 1 hr. Cells were washed with PBS 3X at 5 min each Cells were incubated with Goat anti-Mouse IgG(H+L) conjugated with HRP (1:200) for 30 min at RT. Colormetric signal was developed using DAB solutions.
VALIDATION REPORT: mCherry Antibody (STJ34373)
BACKGROUND
Antibody: mCherry AntibodyMethod of Validation: Immunohistochemistry Validator: Microstem IncSupplier Catalog Number: STJ34373Supplier: St John’s Laboratory Experimental Notes: Strong signal was detected in the positive control, but not in the negative control.
INDEPENDENT VALIDATION METHODS
Primary Antibody: mCherry AntibodyIsotype Control Antibody: mouse IgG1; supplier: CusaBioSecondary Antibody: Goat anti-Mouse IgG(H+L) conjugated with HRP; supplier: KPL
mCherrry antibody has been independently validated for Immunohistochemistry by Microstem Inc.
Figure 1: HEK293 cell transfected with mCherry vector stained with mCherry antibody developed using IHC method.
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VALIDATION REPORT: CD133 Antibody (STJ20168)
BACKGROUND
Antibody: CD133 Antibody Method of Validation: Immunohistochemistry Supplier Catalog Number: STJ20168Supplier: St John’s Laboratory Experimental Notes: Very satisfied! Great communication with seller. The antibody tested stained intensely at the membrane as predicted. Compared to antibodies from other companies, this is the best CD133 staining I have obtained so far.
INDEPENDENT VALIDATION METHODS
Primary Antibody: CD133 Antibody (Dilution: 1:200 in PBS containing 0.1% porcine gelatin)
CD133 antibody has been independently validated for Immunohistochemistry by Tago Santos from Health Sciences Research Center.
Figure 1: Immunocytochemistry on human endothelial progenitor cells isolated from stroke patients. Images acquired by confocal microscopy.
VALIDATION REPORT: RARA Antibody (STJ26160)
BACKGROUND
Antibody: RARA AntibodyMethod of Validation: Western BlotSupplier Catalog Number: STJ26160Supplier: St John’s Laboratory Experimental Notes: The antibody tested gave a clear band at the predicted molecular weight.
INDEPENDENT VALIDATION METHODS
Primary Antibody: RARA Antibody (Dilution: 1:1000 in TBS containing 0.1% porcine gelatin)
RARA antibody has been independently validated for Western Blot by Tago Santos from Health Sciences Research Center.
Figure 1: Western blotting on subventricular zone neural stem cells isolated from mice (primary culture) using ECL substrate.
PROTOCOL
Immunoblotting
After running SDS-gel and transferring proteins to nitrocellulose membrane, such steps were performed:
Blocking with 1X PBS-T containing 5% nonfat dry milk with constant shacking for 1 hour.
Incubation with primary antibody (dilution 1:1000 in blocking buffer) overnight at 4°C.
Washing the membrane with PBS-T 3x10 min.
Incubation with HRP-conjugated secondary antibody for 1h in room temperature.
Washing the membrane with PBS-T 3x10 min.
Imaging of the membrane.
VALIDATION REPORT: LPCAT1 Antibody (STJ27005)
BACKGROUND
Antibody: LPCAT1 AntibodyMethod of Validation: Western Blot Supplier Catalog Number: STJ27005Supplier: St John’s Laboratory Experimental Notes: Product worked as expected.
INDEPENDENT VALIDATION METHODS
Primary Antibody: LPCAT1 Antibody (Dilution: 1:1000)
LPCAT1 antibody has been independently validated for Western Blot by Kristina Klizaite from Life & Medical Sciences University of Bonn.
Figure 1: Western blot image of mouse lung tissue.
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PROTOCOL
Gel electrophoresis: 10% polyacrylamide gel, constant voltage 160 V, 60 min.Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.Blocking: 1X TBST with 5% skimmed milk powder for 2 hr.Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4˚C for overnight.Membrane wash: 1X TBST wash for 3 times.Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1：10000 dilution ratio, for 1.5 hr.Membrane wash: 1X TBST wash for 3 times.Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).
VALIDATION REPORT: HA-Tag Polyclonal Antibody (STJ90107)
BACKGROUND
Antibody: HA-Tag AntibodyMethod of Validation: Western Blot Supplier Catalog Number: STJ90107Supplier: St John’s Laboratory Experimental Notes: In Hela and Jurkat cell line, there were target bands detected by STJ90107. HA-tag Polyclonal Antibody.
INDEPENDENT VALIDATION METHODS
Primary Antibody: HA-Tag Antibody (Dilution 1:10000)Secondary Antibody: HRP goat anti-rabbit (provided by BPI)Reference Antibody: Actin (provided by BPI)
HA-Tag antibody has been independently validated for Western Blot by BPI
Figure 1: Western blot;Hela and Jurkat cell line target bands detected by HA Tag antibody.
Gel electrophoresis infromation：10% polyacrylamide gel, constant voltage 160 V, 60 min.
Transfer information：0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min.
No. Antigen Loading amount Primary antibodyPrimary antibody dilution ratio
Secondaryantibody dilution ratio
Target band KD Visualization time
1 Hela lysate 10 ug St J90107. HA-tag Polyclonal Antibody
1:10001:10000 68KD 34S
2 Jurkat lysate 10 ug 1:10000 68KD 34S
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