Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: USP22 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HEK293 whole cell lysate (20 µg)
Lane 4: HeLa whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab195289 observed at 60 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab195289 was shown to specifically react with USP22 in wild-type HAP1 cells. No band was observed when knockout samples were examined. Wild-type and USP22 knockout samples were subjected to SDS-PAGE. Ab195289 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at a 1/2000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling USP22 with ab195289 at 1/1000 followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Nuclear staining on rat cerebrum. The section was incubated with ab195289 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling USP22 with ab195289 at 1/1000 followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Nuclear staining on mouse cerebrum. The section was incubated with ab195289 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling USP22 with ab195289 at 1/100 followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution. Nuclear staining on human breast carcinoma. The section was incubated with ab195289 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use.

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

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling USP22 with ab195289 at 1/100 followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Nuclear staining on human cerebrum. The section was incubated with ab195289 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use.

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

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: USP22 knockout HAP1 cell lysate (20 μg)
Lane 3: HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate (20 µg)

ab195289 was shown to specifically recognize USP22  when USP22 knockout samples were used. Wild-type and knockout samples were subjected to SDS-PAGE. ab195289 (1/2000) and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.  

All lanes : Anti-USP22 antibody [EPR18945] (ab195289) at 1/2000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-USP22 antibody [EPR18945] (ab195289) at 1/2000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 4 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 5 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 6 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate
Lane 7 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1, 2, 3, 4, 5 and 6: 30 seconds; Lane 7: 15 seconds.

All lanes : Anti-USP22 antibody [EPR18945] (ab195289) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat brain lysate
Lane 4 : Rat spleen lysate
Lane 5 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 6 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 7 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 8 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1, 2, 3 and 4: 3 minutes; Lane 5, 6, 7 and 8: 30 seconds.

 

USP22 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab195289 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab195289 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate, 10µg (Input).

Lane 2: ab195289 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A]- Isotype Control (ab172730) instead of ab195289 in HeLa whole cell lysate.

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

Exposure time: 3 seconds.