重组Anti-FXR1抗体[EPR7932]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FXR1 antibody [EPR7932] (AB129089)

ab129089 staining FXR1 in the human cell line HeLa (human cervix adenocarcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permiabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/60. 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)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FXR1 antibody [EPR7932] (AB129089)

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling FXR1 with purified ab129089 at 1/500 dilution. Cells were fixed with 100% methanol. ab150077 Goat anti rabbit IgG (Alexa Fluor^®488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FXR1 antibody [EPR7932] (AB129089)

ab129089, at a dilution of 1/100, staining FXR1 in paraffin-embedded Human brain tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-FXR1 antibody [EPR7932] (AB129089)

Lanes 1 - 5 : Merged signal (red and green). Green - ab129089 observed at 70-80 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab129089 was shown to react with FXR1 in wild-type HeLa cells in Western blot with loss of signal observed in FXR1 knockout cell line ab264017 (knockout cell lysate ab264505). Wild-type HeLa and FXR1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab129089 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-FXR1 antibody [EPR7932] (ab129089) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

FXR1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human FXR1 knockout HeLa cell line (<a href='/products/cell-lines/human-fxr1-knockout-hela-cell-line-ab264017'>ab264017</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Lane 5:

HEK-293 cell lysate at 20 µg

Predicted band size: 70 kDa

Observed band size: 70-80 kDa

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• WB

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Western blot - Anti-FXR1 antibody [EPR7932] (AB129089)

All lanes:

Western blot - Anti-FXR1 antibody [EPR7932] (ab129089) at 1/1000 dilution

Lane 1:

Fetal muscle lysate at 10 µg

Lane 2:

MCF7 lysate at 10 µg

Lane 3:

293T lysate at 10 µg

Lane 4:

HeLa lysate at 10 µg

Lane 5:

Fetal heart lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 70 kDa

Observed band size: 70-78 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-FXR1 antibody [EPR7932] (AB129089)

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.