All lanes : Anti-HLA Class 1 ABC antibody [EPR22172] (ab225636) at 1/1000 dilution

Lane 1 : Recombinant human HLA-A protein (aa24-aa365) 10ng
Lane 3 : Recombinant human HLA-C protein (aa25-aa308) 10ng

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 40 kDa

This antibody has cross reaction with human HLA-A and HLA-C.# Recombinant human HLA-A protein was purchased from abnova company (H00003105-P01).

Recombinant human HLA-C protein was purchased from novusbio company (NBP2-23210).

Immunofluorescent analysis of 100% methanol-fixed THP-1 (human monocytic leukemia cell line) cells labeling HLAB with ab225636 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in THP-1 cells. The nuclear counterstain is DAPI (blue).
Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).
The negative control is the secondary antibody only.

Methanol is recommended for fixation as weak signal was observed using 4% PFA.

Flow cytometric analysis of Raji (human Burkitt's lymphoma cell line) cells labeling HLAB with ab225636 at 1/600 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti-rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling HLAB with ab225636 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining on lymph nodule of human stomach (PMID:25294906) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-HLA Class 1 ABC antibody [EPR22172] (ab225636) at 1/1000 dilution

Lane 1 : Raji (human Burkitt's lymphoma cell line), whole cell lysate
Lane 2 : THP-1 (human monocytic leukemia cell line), whole cell lysate
Lane 3 : HL-60 (human promyelocytic leukemia cell line), whole cell lysate
Lane 4 : Jurkat (human T cell leukemia cell line from peripheral blood), whole cell lysate
Lane 5 : HepG2 (human liver hepatocellular carcinoma cell line), whole cell lysate
Lane 6 : TF-1 (human bone marrow erythroleukemia cell line), whole cell lysate
Lane 7 : Ramos (human Burkitt's lymphoma cell line), whole cell lysate
Lane 8 : Human tonsil

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 40 kDa
Observed band size: 40 kDa


Exposure time: 26 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Weak reactive bands of undetermined proteins are observed in addition to the target band.

 

HLAB was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia cell line) whole cell lysate with ab225636 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab225636 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.
Lane 1: THP-1 whole cell lysate 10 µg (Input).
Lane 2: ab225636 IP in THP-1 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab225636 in THP-1 whole cell lysate.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 seconds.

Immunofluorescent analysis of 100% methanol-fixed Raji (human Burkitt's lymphoma cell line) cells labeling HLAB with ab225636 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in Raji cells. The nuclear counterstain is DAPI (blue).
Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).
The negative control is the secondary antibody only.

Methanol is recommended for fixation as weak signal was observed using 4% PFA.

Flow cytometric analysis of THP-1 (human monocytic leukemia cell line) cell line labeling HLAB with ab225636 at 1/600 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling HLAB with ab225636 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on hepatic sinus endothelium of human liver (PMID: 26963506) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human colonic carcinoma tissue labeling HLAB with ab225636 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining on tumor cells of human colonic carcinoma (PMID: 26310568, PMID: 17014712) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).