Anti-LRP1 antibody [EPR3724] - BSA and Azide free

• IP

Lab

Immunoprecipitation - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

This data was developed using ab92544, the same antibody clone in a different buffer formulation.

Immunoprecipitation of LRP1 in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2.0 µg of ab92544 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-LRP1 antibody [EPR3724] (<a href='/en-us/products/primary-antibodies/lrp1-antibody-epr3724-ab92544'>ab92544</a>) at 2 µg

All lanes:

HAP1 cells

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• WB

Lab

Western blot - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

This data was developed using ab92544, the same antibody clone in a different buffer formulation.

ab92544 was shown to react with LRP1 in wild-type HAP1 cells in Western blot with loss of signal observed in a LRP1 knockout cell line. Wild-type HAP1 and LRP1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab92544 overnight at 4 °C at a 1/50000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-LRP1 antibody [EPR3724] (<a href='/en-us/products/primary-antibodies/lrp1-antibody-epr3724-ab92544'>ab92544</a>) at 1/50000 dilution

Lane 1:

Wild-type HAP1 lysate at 30 µg

Lane 2:

LRP1 knock-out HAP1 lysate at 30 µg

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• ICC/IF

AbReview33110****

Immunocytochemistry/ Immunofluorescence - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

This image is courtesy of an Abreview submitted by Ruma Raha-Chowdhury.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

This IHC data was generated using the same anti-LRP1 antibody clone, EPR3724, in a different buffer formulation (cat# ab92544).

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 Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

Intracellular 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

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

• IP

Unknown

Immunoprecipitation - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92544).

All lanes:

Immunoprecipitation - Anti-LRP1 antibody [EPR3724] (<a href='/en-us/products/primary-antibodies/lrp1-antibody-epr3724-ab92544'>ab92544</a>)

Predicted band size: 504 kDa

Observed band size: 85 kDa

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• WB

Unknown

Western blot - Anti-LRP1 antibody [EPR3724] - BSA and Azide free (AB215997)

This WB data was generated using the same anti-LRP1 antibody clone, EPR3724, in a different buffer formulation (cat# 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.

All lanes:

Western blot - Anti-LRP1 antibody [EPR3724] (<a href='/en-us/products/primary-antibodies/lrp1-antibody-epr3724-ab92544'>ab92544</a>)

Predicted band size: 504 kDa

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