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Recombinant fragment (Human), 85kDa cleaved fragment of Low Density LRP
Our Abpromise guarantee covers the use of ab28320 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.|
|WB||Use a concentration of 1 µg/ml. Use under non reducing condition. Detects a band of approximately 80 kDa (predicted molecular weight: 85 kDa).
Abcam do not recommend adding a reducing agent (e.g. DTT) to lysates or heating samples prior to loading on SDS-PAGE gels. We have used BSA as our blocking agent at 1%.
|IHC-P||Use a concentration of 10 µg/ml.|
|IHC-Fr||Use at an assay dependent concentration.|
|ELISA||Use at an assay dependent concentration.|
|ICC/IF||Use at an assay dependent concentration.|
This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 1% Bovine Serum Albumin before being incubated with ab28320 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution. Please note that no reducing agent was added to the cell lysate and the sample was not heated prior to loading onto the SDS-PAGE gel.
IHC image of LRP1 staining in Human normal placenta formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with EDTA buffer (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab28320, 10µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Overlay histogram showing HepG2 cells stained with ab28320 (red line). The cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab28320, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a significantly decreased signal in HepG2 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.
Please note that Abcam does not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.
This image is courtesy of an Abreview submitted by Dr Torry Tucker
50µg of human fibroblast whole cell lysate were loaded on to the gel and run under denaturing conditions.
The membrane was blocked with 1% BSA for 1 hour at RT.
The primary antibody was incubated for 12 hours at 4°C.