All lanes : Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101) at 1/5000 dilution

Lane 1 : Untreated Jurkat cells whole cell lysates
Lane 2 : Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates
Lane 3 : Jurkat cells were treated with 50mM Pervanadate for 5 minutes whole cell lysates. Then the membrane was incubated with Alkaline phosphatase.

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 130 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking and diluting buffer 5% NFDM/TBST

Immunocytochemistry/Immunofluorescence analysis of Jurkat +/- pervanadate (1mM, 30min) and Jurkat + pervanadate (1mM, 30min) + LP cells labelling JAK2 (phospho Y1007 + Y1008) with ab32101 at a dilution of 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 (goat anti-rabbit IgG Alexa Fluor^® 488) (1/1000) was used as the secondary antibody. The cells were co-stained with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at a 1/200 dilution. Nuclei counterstained with DAPI (blue).

ab32101 showing positive staining in human lung adenocarcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Dot blot analysis of human JAK2 (phospho Y1007 & Y1008) phospho peptide (Lane 1), JAK2 (phospho Y1007) phospho peptide (Lane 2), JAK2 (phospho Y1008) phospho peptide (Lane 3) and JAK2 non-phospho peptide (Lane 4) labelling JAK2 (phospho Y1007 & Y1008) with ab32101 at a dilution of 1/1000.

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101) at 1/1000 dilution

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates
Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) treated with 50mM Pervanadate for 5 minutes whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 130 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?
Additional bands at: 60 kDa. We are unsure as to the identity of these extra bands.

Blocking and diluting buffer: 5% NFDM/TBST

Exposure time: 
Left image: 1 second
Right image: 5 minutes

Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells starved of serum for 16 hours then treated with 1 mM Pervanadate for 30 minutes labeling JAK2 (phospho Y1007 + Y1008) with ab32101 at 1/20 dilution (10 ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control. Unstimulated Jurkat cells were used as a negative control (Green). 

Direct ELISA antigen dose-response curve using ab32101 at 0~1000 ng/mL. Antigen concentration of 100 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (1/2500) was used as the secondary antibody.

This antibody preferentially recognizes phospho Y1007. When the concentration of peptides is higher than 100 ng/mL, it also recognizes phospho Y1008.

Direct ELISA antigen dose-response curve using ab32101 at 0~1000 ng/mL. Antigen concentration of 10 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (1/2500) was used as the secondary antibody.

This antibody preferentially recognizes phospho Y1007. When the concentration of peptides is lower than 10 ng/mL, it cannot recognize phospho Y1008.

 

Analysis of immunogen phosphopeptide (human JAK2 (phospho Y1007 + Y1008)) (A) and non phosphopeptide (B). Antibody used at 1/1000 dilution.

All lanes : Anti-JAK2 (phospho Y1007 + Y1008) antibody [E132] (ab32101) at 1/2000 dilution

Lane 1 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 0 hours.
Lane 2 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 15 minutes.
Lane 3 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 30 minutes.
Lane 4 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 1 hour.
Lane 5 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 2 hours.
Lane 6 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 4 hours.
Lane 7 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 6 hours.
Lane 8 : Rat uterine cell line - whole cell lysate. Treated with 1µg/mL Prolactin for 24 hours.

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : An HRP-conjugated donkey anti-rabbit polyclonal. at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 130 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?
Additional bands at: 55 kDa (possible non-specific binding)

See Abreview

ab32101 showing positive staining in human ovarian carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab32101 showing positive staining in human cervical carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab32101 showing positive staining in human endometrial carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab32101 showing positive staining in human urinary bladder transitional carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.