700422

antibody from Invitrogen Antibodies

Targeting: HK2

Provider product page for 700422

Western blot

Immunocytochemistry

Flow cytometry

Antibody data

Antibody Data
Antigen structure
References [0]
Comments [0]
Validations
Western blot [5]
Immunocytochemistry [3]
Flow cytometry [1]

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Product number: 700422 - Provider product page
Provider: Invitrogen Antibodies
Product name: HK2 Recombinant Rabbit Monoclonal Antibody (2H8L6)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: Immunogen sequence: MAEKVLVTG GAGYIGSHTV LELLEAGYLP VVIDNFHNAF RGGGSLPESL RRVQELTGRS VEFEEMDILD QGALQRLFKK YSFMAVIHFA GLKAVGESVQ KPLDYYRVNL TGTIQLLEIM KAHGVKNLVF SSSATVYGNP QYLPLDEAHP TGGCTNPYGK SKFFIEEMIR DLCQADKTWN AVLLRYFNPT GAHASGCIGE DPQGIPNNLM PYVSQVAIGR REALNVFGND YDTEDGTGVR DYIHVVDLAK GHIAALRKLK EQCGCRIYNL GTGTGYSVLQ MVQAMEKASG KKIPYKVVAR REGDVAACYA NPSLAQEELG WTAALGLDRM CEDLWRWQKQ NPSGFGTQA (1-348 aa encoded by BC050685 )
Reactivity: Human
Host: Rabbit
Isotype: IgG
Antibody clone number: 2H8L6
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

HK2 protein structure - 700422 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HCT 116 (Lane 1), HT -29 (lane 2), SK-O-V3 (lane 3), T47D (Lane 4), MDA-MB-231 (lane 5), and MCF-7 (Lane 6).The blots were probed with Anti-Hexokinase II Rabbit Monoclonal Antibody (Product # 700422, 3 µg/mL) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A 102kDa band corresponding to Hexokinase II was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with Pierce™ Power Blotter System (Product # 22834). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Hexokinase-2 was achieved by transfecting U-87 MG with Hexokinase-2 specific siRNAs (Silencer® select Product # s6560, s6562). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Hexokinase-2 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HK2 Recombinant Rabbit Monoclonal Antibody (2H8L6) (Product # 700422, 2 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Hexokinase-2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-HK2 Recombinant Rabbit Monoclonal Antibody (2H8L6) (Product # 700422) and a 102 kDa band corresponding to Hexokinase-2 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), HCT 116 (Lane 2), Caco-2 (Lane 3), and HeLa (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of HK2 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1034965_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of HK2 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) andHeLa HK2 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-HK2 Recombinant Rabbit Monoclonal Antibody (2H8L6) (Product # 700422, 3 µg/mL dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:6000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to HK2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Hexokinase 2 was performed by loading 20 µg of HCT116 cell lysate (lane 1) and MiaPaCa-2 lysate (lane 2) in reducing sample buffer and Page Rµler Protein Ladder (Product # 26616) onto a 10% polyacrylamide gel. Proteins were transferred to PVDF membrane using the wet transfer method. Membrane was blocked in 5% milk. Hexokinase 2 was detected at approximately 102 kDa using a Hexokinase 2 monoclonal antibody (Product # 700422) at 3 µg/mL, followed by a goat anti-rabbit secondary antibody at a dilution of 1:7500 in 5% milk, 0.01% SDS. Data courtesy of Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Hexokinase II was done on 70% confluent log phase MCF-7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Hexokinase II (2H8L6), Recombinant Rabbit Monoclonal Antibody (Product # 700422) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Hexokinase-2 was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.01% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with HK2 Recombinant Rabbit Monoclonal Antibody (2H8L6) (Product # 700422) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al. / Methods 115 (2017) 28-41).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Hexokinase-2 was performed using 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.01% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with HK2 Recombinant Rabbit Monoclonal Antibody (2H8L6) (Product # 700422) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2,000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al. / Methods 115 (2017) 28–41).