Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Hsp60 with ab190828 at 1/200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells.

The nuclear counter stain is DAPI (blue). COX IV is detected with Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) (red) at 1/1000 dilution.

The negative controls are as follows : -

-ve control 1 : ab190828 at 1/200 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) at 1/1000 dilution.

-ve control 2 : Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Hsp60 with ab190828 at 1/200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Hsp60 with ab190828 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granular and cytoplasmic staining on human liver (PMID : 18548335). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embyro fibroblast cell line) cell line labeling Hsp60 with ab190828 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embyro fibroblast cell line) cells labeling Hsp60 with ab190828 at 1/200 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Hsp60 with ab190828 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granular and cytoplasmic staining on rat liver (PMID : 18548335). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Hsp60 was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embyro fibroblast cell line) cell lysate with ab190828 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab190828 at 1/5000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : NIH/3T3 cell lysate 10 μg (Input).

Lane 2 : ab190828 IP in NIH/3T3 cell lysate .

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab190828 in NIH/3T3 cell lysate .

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (ab190828)

Predicted band size: 61 kDa

false

• WB

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Western blot - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Exposure time : Lanes 1,2 and 3 : 3 minutes; Lane 4 : 1 minute.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (ab190828) at 1/1000 dilution

Lane 1:

Human brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 61 kDa

Observed band size: 61 kDa

true

• WB

Supplier Data

Western blot - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Exposure time : Lanes 1,2 and 3 : 30 seconds; Lane 4 : 15 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (ab190828) at 1/5000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embyro fibroblast cell line) cell lysate at 20 µg

Lane 3:

Neuro-2a (mouse neuroblastoma cell line) cell lysate at 20 µg

Lane 4:

C6 (rat glial tumor cell line) cell lysate at 20 µg

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 1/100000 dilution

Predicted band size: 61 kDa

Observed band size: 61 kDa

true

• WB

Supplier Data

Western blot - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (AB190828)

Exposure time : Lane 1 : 30 seconds; Lane 2 : 1 minute; Lanes 3,4 and 5 : 10 seconds; Lane 6 : 5 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Hsp60 antibody [EPR18245-93] - Mitochondrial Marker (ab190828) at 1/1000 dilution

Lane 1:

Mouse heart lysate at 10 µg

Lane 2:

Mouse spleen lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

Rat heart lysate at 10 µg

Lane 5:

Rat spleen lysate at 10 µg

Lane 6:

Rat liver lysate at 10 µg

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 1/100000 dilution

Predicted band size: 61 kDa

Observed band size: 61 kDa

true