Anti-CLPTM1 antibody [EPR8800]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CLPTM1 antibody [EPR8800] (AB133756)

Flow Cytometry analysis of A549 (Human lung carcinoma epithelial cell) cells labeling CLPTM1 with purified ab133756 at 1/200 dilution (1 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CLPTM1 antibody [EPR8800] (AB133756)

Immunofluorescent staining of HeLa cells labelling CLPTM1 with ab133756 at 1/250.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLPTM1 antibody [EPR8800] (AB133756)

Immunohistochemical analysis of paraffin embedded papillary adenocarcinoma of Human thyroid gland tissue labelling CLPTM1 with ab133756 at 1/50.

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

• WB

Lab

Western blot - Anti-CLPTM1 antibody [EPR8800] (AB133756)

Lanes 1 - 4 : Merged signal (red and green). Green - ab133756 observed at 76 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133756 was shown to recognize in wild-type HAP1 cells as signal was lost at the expected MW in CLPTM1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CLPTM1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3pc Milk. ab133756 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CLPTM1 antibody [EPR8800] (ab133756) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CLPTM1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

A549 whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Predicted band size: 76 kDa

Observed band size: 76 kDa

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• WB

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Western blot - Anti-CLPTM1 antibody [EPR8800] (AB133756)

All lanes:

Western blot - Anti-CLPTM1 antibody [EPR8800] (ab133756) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

U87 MG cell lysate at 10 µg

Lane 4:

HepG2 cell lysate at 10 µg

Lane 5:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 76 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-CLPTM1 antibody [EPR8800] (AB133756)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.