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.

Coronal sections of the E13.5 (a), E15.5 (c) and E18 (g)mouse brain and transverse sections of the E15.5 spinal cord (e) were immunolabelled to detect radial glia (RC2, green) and LRP1 (red). LRP1 was detected using ab92544 at 1/500 dilution. sections were cut to 30μm and collected as floating sections into PBS. Cryosections were exposed to primary antibodies diluted in blocking solution [10% (v/v) fetal calf serum and 0.1% (v/v) triton x100 in PBS] and incubated overnight at 4°C on an orbital shaker.

The nuclear marker Hoechst 33342 was used to label cell nuclei (blue). (b,d,f,h,j) secondary antibody alone controls. All images are single z plane confocal scans. White arrows indicate regions of co-localisation. Scale bars represent 17μm. SC = spinal cord.

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.

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

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

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.

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).

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.

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

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.

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

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

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