All lanes : Anti-67kDa Laminin Receptor antibody [EPR8469] (ab133645) at 1/10000 dilution

Lane 1 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 33 kDa

Blocking and diluting buffer: 5% NFDM/TBST

ab133645 staining Prohibitin in mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab133645 staining 67kDa Laminin Receptor in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a counterstain for primary antibody ab133645 at 1/2000. DAPI was used as a nuclear counterstain and PBS as a negative control.

ab133645 staining 67kDa Laminin Receptor in the human cell line MCF7 (Human breast adenocarcinoma cell line) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

ab133645 immunoprecipitating 67kDa Laminin Receptor. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/20 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/1000.

Lane 1: K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate (10ug)
Lane 2: K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab133645 in K562(Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate

ab133645 staining 67kDa Laminin Receptor in rat kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab133645 staining 67kDa Laminin Receptor in mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab133645 staining 67kDa Laminin Receptor in human gastric carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

All lanes : Anti-67kDa Laminin Receptor antibody [EPR8469] (ab133645) at 1/2000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : C6 (Rat glial tumor cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 33 kDa

Diluting and blocking buffer: 5% NFDM/TBST

Overlay histogram showing MCF7 cells stained with ab133645 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab133645, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-67kDa Laminin Receptor antibody [EPR8469] (ab133645) at 1/1000 dilution

Lane 1 : K562 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : C6 cell lysate
Lane 5 : RAW 264.7 cell lysate
Lane 6 : PC-12 cell lysate
Lane 7 : NIH/3T3 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 33 kDa

Immunohistochemical analysis of paraffin-embedded Human breast tissue labelling 67kDa Laminin Receptor with ab133645 at 1/100 dilution.

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

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labellig 67kDa Laminin Receptor with ab133645 at 1/100 dilution.

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

All lanes : Anti-67kDa Laminin Receptor antibody [EPR8469] (ab133645) at 1/1000 dilution

All lanes : Mouse intestinal mucosa whole tissue lysate

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG polyclonal at 1/2500 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

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