Anti-DNAJC5B antibody [EPR24152-62]

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

IHC image of DNAJC5A+5B staining in a section of frozen normal human cerebellum* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab259836, 0.5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labelling DNAJC5A+5B with ab259867 at 1/300 (558.0 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human cerebrum. The section was incubated with ab259867 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

DNAJC5A+5B was immunoprecipitated from 0.35 mg Human cerebellum tissue lysate 10 ug with ab259867 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259867 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Human cerebellum tissue lysate 10 ug

Lane 2 : ab259867 IP in Human cerebellum tissue lysate

Lane 3 : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated : Rabbit monoclonal IgG (ab172730) instead of ab259867 in human cerebellum tissue lysate

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

Exposure time : 7.5 seconds

All lanes:

Immunoprecipitation - Anti-DNAJC5B antibody [EPR24152-62] (ab259867)

Predicted band size: 23 kDa

Observed band size: 22-30 kDa

false

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat cerebral cortex tissue labeling DNAJC5A+5B with ab259867 at 1/100 (5.58 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Cytoplasmic staining on rat cerebral cortex is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling DNAJC5A+5B with ab259867 at 1/300 (558.0 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on mouse cerebrum. The section was incubated with ab259867 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt

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Flow Cytometry - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

Flow cytometric analysis of Mouse primary neuron cells cells labelling DNAJC5A+5B with ab259867 at 1/50 dilution (1ug)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. Goat anti rabbit IgG (Alexa Fluor® 647, ab150079) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebral cortex tissue labeling DNAJC5A+5B with ab259867 at 1/100 (5.58 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Cytoplasmic staining on mouse cerebral cortex is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling DNAJC5A+5B with ab259867 at 1/300 (558.0 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on rat cerebrum. The section was incubated with ab259867 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

Lab

Western blot - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

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

DNAJC5A has non-palmitoylated and palmitoylated forms as described in the literature (PMID : 32042150).

Low expression : kidney (PMID : 20499929).

Exposure time : 26 seconds

All lanes:

Western blot - Anti-DNAJC5B antibody [EPR24152-62] (ab259867) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Human kidney tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue lysate at 20 µg

Lane 5:

Mouse testis tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

Rat kidney tissue lysate at 20 µg

Lane 8:

Rat testis tissue 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 1/50000 dilution

Predicted band size: 23 kDa

Observed band size: 22-30 kDa

false

• WB

Lab

Western blot - Anti-DNAJC5B antibody [EPR24152-62] (AB259867)

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

This antibody reacts with both DNAJC5A and DNAJC5B.

Exposure time : 37 seconds

All lanes:

Western blot - Anti-DNAJC5B antibody [EPR24152-62] (ab259867) at 1/1000 dilution

Lane 1:

His-tagged human DNAJC5/CSP recombinant protein (aa 2-100) at 0.01 µg

Lane 2:

Myc/DDK-tagged human DNAJC5B recombinant protein (aa 1-199) at 0.02 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: 23 kDa

false