Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling Hexokinase 1 with Purified ab150423 at 1/20 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488 ,ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling Hexokinase 1 with purified ab150423 at 1/50 dilution (4.14 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Fluorescence multiplex immunohistochemical analysis of the human kidney (Formalin/PFA-fixed paraffin-embedded sections). Panel A : merged staining of anti-Hexokinase 1 (ab150423, green; Opal™690), anti-Angiotensin Converting Enzyme 1 (ab254222, gray; Opal™520) and anti-Aquaporin 2 (ab199975, red; Opal™570) on human kidney. Panel B : anti-Aquaporin 2 stained on collecting tubules. Panel C : anti-Angiotensin Converting Enzyme 1 stained on proximal tubules. Panel D : anti-Hexokinase 1 stained on distal tubules and collecting tubules. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The section was incubated in three rounds of staining : in the order of ab150423 at 1/250 dilution (4.224 μg/ml), ab254222 at 1/4000 dilution (0.141 μg/ml) and ab199975 at 1/4000 dilution (0.152 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Immunocytochemistry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Hexokinase 1 with Purified ab150423 at 1 : 50 dilution (4.1 µg/ml). Cells were fixed in 100% Methanol and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488,ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Fluorescence multiplex immunohistochemical analysis of paraffin-embedded Human kidney tissue. Panel A : Merged staining of anti-Hexokinase 1 (gray; Opal™690), anti-Angiotensin Converting Enzyme 1 (green; Opal™520) and anti-Tissue Factor (red; Opal™570) on human kidney. Panel B : Anti-Tissue Factor stained on renal glomeruli. Panel C : Anti-Angiotensin Converting Enzyme 1 stained on proximal tubules. Panel D : Anti-Hexokinase 1 stained on distal tubules. The section was incubated in three rounds of staining : in the order of ab150423, ab254222, and ab252918 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Counterstained with DAPI.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat kidney tissue sections labeling Hexokinase 1 with purified ab150423 at 1/50 dilution (4.14 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse kidney tissue sections labeling Hexokinase 1 with purified ab150423 at 1/50 dilution (4.14 µg/mL). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Unknown

Western blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Lanes 1-4 : Merged signal (red and green). Green - ab150423 observed at 102 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab150423 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial was shown to specifically react with Hexokinase 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267279 (knockout cell lysate ab257161) was used. Wild-type and Hexokinase 1 knockout samples were subjected to SDS-PAGE. ab150423 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (ab150423) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

HK1 knockout HEK293T cell lysate at 20 µg

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Predicted band size: 102 kDa

Observed band size: 102 kDa

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• WB

Lab

Western blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Lanes 1-4 : Merged signal (red and green). Green - ab150423 observed at 102 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab150423 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial was shown to specifically react with Hexokinase 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267279 (knockout cell lysate ab257161) was used. Wild-type and Hexokinase 1 knockout samples were subjected to SDS-PAGE. ab150423 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (ab150423) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HK1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human HK1 (Hexokinase 1) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hk1-hexokinase-1-knockout-hek-293t-cell-line-ab267279'>ab267279</a>)

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

HEPG2 cell lysate at 20 µg

Predicted band size: 102 kDa

Observed band size: 102 kDa

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• WB

Unknown

Western blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

All lanes:

Western blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (ab150423) at 1/1000 dilution

Lane 1:

Human brain lysate

Lane 2:

Mouse brain lysate

Lane 3:

Rat brain lysate

Lane 4:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate

Lane 5:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 102 kDa

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• Dot

Supplier Data

Dot Blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Dot blot analysis of Hexokinase Type III/HK3 using ab321996 at 1 : 1000 (0.525 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : His-tagged human HK3 fragment
Lane2 : His-tagged human HK2 fragment
Lane3 : His-tagged human HK1 fragment
Lane4 : His-tagged human HKDC1 fragment

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with human HK1, HK2 and HKDC1.

All lanes:

Dot Blot - Anti-Hexokinase Type III/HK3 antibody [EPR29196-29] (<a href='/en-us/products/primary-antibodies/hexokinase-type-iii-hk3-antibody-epr29196-29-ab321996'>ab321996</a>) at 1/1000 dilution

Lane 1:

His-tagged human HK3 fragment at 5 ng

Lane 2:

His-tagged human HK2 fragment at 5 ng

Lane 3:

His-tagged human HK1 fragment at 5 ng

Lane 4:

His-tagged human HKDC1 fragment at 5 ng

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

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Exposure time: 180s

• WB

CiteAb

Western blot - Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB150423)

Hexokinase 1 western blot using anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker ab150423. Publication image and figure legend from Dijk, S. N., Protasoni, M., et al., 2020, Sci Rep, PubMed 32152409.

ab150423 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab150423 please see the product overview.

Doxycycline inhibits mitochondrial translation. (a) De novo mitochondrial protein synthesis of fibroblasts (Fibs), A549 and A549 ρ° cells treated with vehicle (-) or doxycycline (+). After 5 days of treatment, cultures were pulse-labelled with [35S]-methionine in the presence of emetine. Protein samples were subjected to gel electrophoresis. The gel was stained with Coomassie brilliant blue followed by autoradiography to reveal the labelled mitochondrial translation products, indicated on the left. The migration of protein standards is indicated in the centre. (b) Mean MTCO2 + MTCO3 labelling signals in vehicle (Veh) and doxycycline (DC)-treated cells relative to the mean value of vehicle-treated A549 cells (n = 4). (c) Western blot images of samples from fibroblasts and A549 cells treated with doxycycline over a 5-day period and from untreated A549 ρ° cells. To facilitate quantification, serial dilutions of untreated cells, harvested at t = 0, were also applied. Blots were probed with antibodies against the indicated proteins. Migration of protein standards is indicated on the right. Full-size blots are presented in Supplementary Fig. S11. (d) Mean amounts of the indicated proteins in the treated cells over a 5-day period and of untreated A549 ρ° cells, relative to the amount in the cells at t = 0 (n = 3). Error bars indicate standard deviations. Asterisks denote statistically significant differences (p < 0.05).

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