Anti-M6PR (cation independent) 抗体 [EPR6599]

• WB

Lab

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : M6PR (cation independent) knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : A549 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab124767 observed at 274 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab124767 was shown to specifically react with M6PR (cation independent) in wild-type HAP1 cells as signal was lost in M6PR (cation independent) knockout cells. Wild-type and M6PR (cation independent) knockout samples were subjected to SDS-PAGE. ab124767 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/50000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Lane 1:

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (ab124767)

Lanes 2 - 4:

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - BSA and Azide free (<a href='/products/primary-antibodies/m6pr-cation-independent-antibody-epr6599-bsa-and-azide-free-ab226090'>ab226090</a>) at 1/50000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

M6PR (cation independent) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

A549 whole cell lysate at 20 µg

Predicted band size: 274 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Immunohistochemical analysis of formalin fixed, paraffin embedded Human tonsil tissue section labelling Mannose 6 Phosphate Receptor (Cation independent) with unpurified ab124767 at dilution of 1/250.

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Immunocytochemistry/immunofluorescence analysis of 293T cells labelling Mannose 6 Phosphate Receptor (Cation independent) with unpurified ab124767 at dilution of 1/100.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

ab124767 staining M6PR in wild-type HAP1 cells (top panel) and IGF2R knockout HAP1 cells (bottom panel). The cells were fixed with 100% MeOH for 5 min. , permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab124767 at 1μg/ml and ab195889 (Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594)) at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Immunohistochemical staining of paraffin embedded human thyroid carcinoma tissue section labelling M6PR with purified ab124767 at dilution of 1/500. The secondary antibody used was ab97051 Goat Anti-Rabbit IgG H&L (HRP), at a dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Immunocytochemistry/Immunofluorescence analysis of Jurkat (human acute T cell leukemia) cells labelling M6PR with purified ab124767 at 1/100. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary. Nuclei couterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used, followed by anti-mouse secondary antibody (ab150120). For negative control 2, mouse primary antibody (ab7291) was used followed by anti-rabbit secondary antibody (ab150077).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labelling M6PR with purified ab124767 at 1/150 (red). Cells were fixed with 4% paraformaldehyde. Alexa Fluorr^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Immunohistochemical analysis of formalin fixed, paraffin embedded Human papillary carcinoma tissue section labelling Mannose 6 Phosphate Receptor (Cation independent) with unpurified ab124767 at dilution of 1/250.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Immunohistochemical staining of paraffin embedded rat colon tissue section labelling M6PR with purified ab124767 at dilution of 1/500. The secondary antibody used was ab97051 Goat Anti-Rabbit IgG H&L (HRP), at a dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Immunohistochemical staining of paraffin embedded mouse colon tissue section labelling M6PR with purified ab124767 at dilution of 1/500. The secondary antibody used was ab97051 Goat Anti-Rabbit IgG H&L (HRP), at a dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• WB

Unknown

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (ab124767) at 1/200000 dilution

Lane 1:

Jurkat (human acute T cell leukemia) whole cell lysate at 20 µg

Lane 2:

HEK293 (human embryonic kidney) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 274 kDa

Observed band size: 300 kDa

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• WB

Unknown

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (ab124767) at 1/50000 dilution

Lane 1:

C6 (rat glioma) whole cell lysate at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Lane 3:

NIH/3T3 (mouse embryo) whole cell lysate at 20 µg

Lane 4:

Mouse heart tissue lysate at 20 µg

Lane 5:

Mouse kidney tissue lysate at 20 µg

Lane 6:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 100 kDa,107 kDa,183 kDa,274 kDa,35 kDa

Observed band size: 120 kDa,300 kDa,43 kDa

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• WB

Unknown

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

All lanes:

Western blot - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (ab124767) at 1/50000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

Caco-2 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-M6PR (cation independent) antibody [EPR6599] - Lysosome Membrane Marker (AB124767)

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.