重组Anti-PMP70抗体[EPR5614]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PMP70 antibody [EPR5614] (AB109448)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling PMP70 with primary antibody anti-PMP70 (ab109448) at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) (ab150081) secondary antibody at 1/1000 dilution (2.0 μg/mL). Confocal image showing cytoplasmic staining on HeLa cells. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (2.5 μg/mL). The nuclear counter stain is DAPI (blue).
The secondary antibody only control is : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution (2.0 μg/mL).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PMP70 antibody [EPR5614] (AB109448)

ab109448 at 1/100 dilution staining PMP70 in permeabilized HepG2 cells by intracellular flow cytometry (shown in red). Rabbit IgG negative control (shown in green).

• WB

Lab

Western blot - Anti-PMP70 antibody [EPR5614] (AB109448)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109448 observed at 75 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab109448 was shown to specifically react with PMP70 in wild-type HAP1 cells as signal was lost in ABCD3 (PMP70) knockout cells. Wild-type and ABCD3 (PMP70) knockout samples were subjected to SDS-PAGE. ab109448 and ab9484 (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-PMP70 antibody [EPR5614] (ab109448) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

ABCD3 (PMP70) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

A431 whole cell lysate at 20 µg

Lane 4:

HEPG2 whole cell lysate at 20 µg

Predicted band size: 75 kDa

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• WB

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Western blot - Anti-PMP70 antibody [EPR5614] (AB109448)

All lanes:

Western blot - Anti-PMP70 antibody [EPR5614] (ab109448) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

SH SY5Y cell lysate at 10 µg

Lane 3:

Caco2 cell lysate at 10 µg

Lane 4:

HepG2 cell lysate at 10 µg

Predicted band size: 75 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-PMP70 antibody [EPR5614] (AB109448)

Lanes 1- 2 : Merged signal (red and green). Green - ab109448 observed at 75 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab109448 was shown to react with PMP70 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265294 (knockout cell lysate ab257134) was used. Wild-type HeLa and ABCD3 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109448 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PMP70 antibody [EPR5614] (ab109448) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 40 µg

Lane 2:

ABCD3 knockout HeLa cell lysate at 40 µg

Predicted band size: 75 kDa

Observed band size: 75 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-PMP70 antibody [EPR5614] (AB109448)

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.