Recombinant Anti-LRP1 antibody [EPR3724] (ab92544)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: LRP1 knockout HAP1 cell lysate (20 µg)
Lane 3: A549 cell lysate (20 µg)
Lane 4: Mouse heart tissue lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab92544 observed at 92 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab92544 was shown to specifically react with LRP1 in wild-type HAP1 cells. No band was observed when LRP1 knockout samples were used. Wild-type and LRP1 knockout samples were subjected to SDS-PAGE. ab92544 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1/10,000 respectively and incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/Immunofluorescence analysis of U87-MG cells labelling LRP1 with purified ab92544 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human clear cell carcinoma of the kidney tissue labelling LRP1 with purified ab92544 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Flow Cytometry analysis of Jurkat cells labelling LRP1 with purified ab92544 at 1/100 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

ab92544 (purified) at 1/30 immunoprecipitating LRP1 in A549 cell lysate. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human brain tissue labelling LRP1 with unpurified ab92544.

ICC/IF image of LRP1 staining on rat mixed glia culture using unpurified ab92544 (1:200). The cells were fixed using paraformaldehyde. The cells were then permeabilised using 0.1% TritonX in 0.1% PBS. Non-specific protein was blocked using 10% donkey serum at 24°C for 1 hour. ab92544 was diluted (1/200) using 0.1% TritonX with 0.1% PBS and 10% donkey serum and the cells were incubated for 4 hours at 24°C. The secondary antibody used was donkey polyclonal to Rabbit IgG conjugated to Alexa Fluor^® 488. DAPI was used to stain the nucleus.

See Abreview

Overlay histogram showing Jurkat cells stained with unpurified ab92544 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab92544, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human lung tissue labelling LRP1 with unpurified ab92544.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human placenta tissue labelling LRP1 with unpurified ab92544.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling LRP1 with unpurified ab92544 at 1/100.