MA5-25174

antibody from Invitrogen Antibodies

Targeting: KHK

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Flow cytometry (Recommended by provider)

Antibody Data

Product number

MA5-25174

Provider

Invitrogen Antibodies

Product name

KHK Monoclonal Antibody (OTI3D1)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Reactivity

Human

Host

Mouse

Isotype

IgG

Antibody clone number

OTI3D1

Vial size

100 µL

Concentration

0.84 mg/mL

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of KHK in HEK293T cells in untransfected (Left lane) and transfected (Right lane) samples using 5 µg per lane. The samples were separated by SDS-PAGE and probed with KHK (Product # MA5-25174) monoclonal antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of KHK in HEK293T cells in untransfected (Left lane) and transfected (Right lane) samples using 5 µg per lane. The samples were separated by SDS-PAGE and probed with KHK (Product # MA5-25174) monoclonal antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of Ketohexokinase was achieved by transfecting Hep G2 with Ketohexokinase specific siRNAs (Silencer® select Product # S7828, S7830). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Ketohexokinase knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with KHK Monoclonal Antibody (OTI3D1) (Product # MA5-25174, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 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 Ketohexokinase.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-KHK Monoclonal Antibody (OTI3D1) (Product # MA5-25174) and a 32 kDa band corresponding to Ketohexokinase was observed along with uncharacterized band (*) across cell lines tested. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), Hep G2 (Lane 2) and Jurkat (Lane 3) 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of KHK in COS7 cells. Cells were transfected with a plasmid overexpressing KHK and probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of KHK was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with KHK Monoclonal Antibody (OTI3D1) (Product # MA5-25174) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), 1:2000 dilution 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 dilution). 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 # CX7C1115LZR).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of KHK was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with KHK Monoclonal Antibody (OTI3D1) (Product # MA5-25174) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), 1:2000 dilution 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 dilution). 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 # CX7C1115LZR).

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human breast tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded carcinoma of human lung tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded carcinoma of human pancreas tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded adenocarcinoma of human ovary tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human lung tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human colon tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded carcinoma of human prostate tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human breast tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded adenocarcinoma of human breast tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded carcinoma of human kidney tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human lymphoma tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded adenocarcinoma of human colon tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded adenocarcinoma of human endometrium tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded carcinoma of human thyroid tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human kidney tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human prostate tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded human liver tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry was performed on paraffin-embedded carcinoma of human bladder tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a KHK monoclonal antibody (Product # MA5-25174).