Anti-M6PR (cation dependent) antibody [EPR7691]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation dependent) antibody [EPR7691] (AB134153)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling M6PR (cation dependent) with Purified ab134153 at 1 : 100 dilution (8.6 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor®594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor®488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-M6PR (cation dependent) antibody [EPR7691] (AB134153)

Intracellular Flow Cytometry analysis of A549 (Human lung carcinoma epithelial cell) cells labeling M6PR (cation dependent) with purified ab134153 at 1/80 dilution (10 μg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

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Western blot - Anti-M6PR (cation dependent) antibody [EPR7691] (AB134153)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-M6PR (cation dependent) antibody [EPR7691] (ab134153) at 0.8 µg/mL

Lane 1:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

Mouse kidney lysates at 15 µg

Lane 3:

Rat kidney lysates at 15 µg

Lane 4:

Rat spleen lysates at 15 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 31 kDa

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• WB

Lab

Western blot - Anti-M6PR (cation dependent) antibody [EPR7691] (AB134153)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : Empty
Lane 3 : M6PR knockout HAP1 whole cell lysate (20 μg)
Lane 4 : A549 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - unpurified ab134153 observed at 46 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab134153 was shown to specifically react with M6PR when M6PR knockout samples were used. Wild-type and M6PR knockout samples were subjected to SDS-PAGE. ab134153 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-M6PR (cation dependent) antibody [EPR7691] (ab134153)

Predicted band size: 31 kDa

Observed band size: 46 kDa

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• WB

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Western blot - Anti-M6PR (cation dependent) antibody [EPR7691] (AB134153)

All lanes:

Western blot - Anti-M6PR (cation dependent) antibody [EPR7691] (ab134153) at 1/1000 dilution

Lane 1:

A549 lysates at 10 µg

Lane 2:

Human uterus lysates at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 31 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-M6PR (cation dependent) antibody [EPR7691] (AB134153)

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.