All lanes : Anti-DNAJC5B antibody [EPR24152-62] (ab259867) at 1/1000 dilution

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Human kidney tissue lysate
Lane 3 : Mouse brain tissue lysate
Lane 4 : Mouse kidney tissue lysate
Lane 5 : Mouse testis tissue lysate
Lane 6 : Rat brain tissue lysate
Lane 7 : Rat kidney tissue lysate
Lane 8 : Rat testis tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 23 kDa
Observed band size: 22-30 kDa why is the actual band size different from the predicted?

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 : 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 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 23 kDa

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

This antibody reacts with both DNAJC5A and DNAJC5B.

Exposure time: 37 seconds

 

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

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

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

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).

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).

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.

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