LRP2 antibody (AA 3401-3500) (Cy3)

Catalog No. ABIN750991

This independently validated Rabbit Polyclonal antibody specifically detects LRP2 in FACS, IF (cc) and IF (p). It exhibits reactivity toward Human and Mouse.

Quick Overview for LRP2 antibody (AA 3401-3500) (Cy3) (ABIN750991)

Target: LRP2 (Low Density Lipoprotein Receptor-Related Protein 2 (LRP2))

Reactivity: Human, Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This LRP2 antibody is conjugated to Cy3

Application: Flow Cytometry (FACS), Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p))

Product Details anti-LRP2 Antibody (Cy3)

Binding Specificity: AA 3401-3500

Cross-Reactivity: Human, Mouse

Predicted Reactivity: Rat,Dog,Horse,Chicken,Rabbit

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic peptide derived from human Megalin

Isotype: IgG

Application Details

Application Notes: FCM 1:20-100
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200

Restrictions: For Research Use only

Independent Validation (IF)

Validation #029611 (Immunofluorescence)

by: CaresBio Laboratory

No.: #029611

Date: 02/11/2014

Lot Number: YEYY9

Method validated: Immunofluorescence

Positive Control: MCF7 cells

Negative Control: HeLa cells

Notes: Strong signal was detected in positive control tissues and not in negative control tissues. Note that there was a small amount of signal generated in the negative control sample.

Validation Images

Figure 1: MCF7 cells stained at 1:250 with LRP2 (red) and a nuclear counterstain (blue).

Figure 2: MCF7 cells stained at 1:100 with LRP2 (red) and a nuclear counterstain (blue).

Figure 3: HeLa cells stained with LRP2 (red) and a nuclear counterstain (blue).

Figure 4: MCF7 cells stained isotype control (red) and a nuclear counterstain (blue).

Figure 1: MCF7 cells with only a secondary antibody (red) and a nuclear counterstain (blue).

Full Methods

Primary Antibody:

• Antigen: Low Density Lipoprotein Receptor-Related Protein 2 (LRP2) antibody (Cy3)
• Catalog number: ABIN750991
• Lot number: YEYY9

Secondary Antibody:

• Used only for the secondary only control, as primary antibody was directly conjugated to Cy3
• Antibody: Cy3 Goat Anti-Rabbit IgG (H+L)

Full Protocol:

• MCF7 and HeLa cell lines were grown directly on coverslips and fixed with 4% formaldehyde in PBS for 15 min at room temperature (RT).
• Fixed cells were rinsed three times in PBS for 5 min each at RT.
• Cells were blocked in 1X PBS/1% BSA/0.3% Triton™ X-100 to block unspecific binding of the antibodies for 60 min at RT.
• Cells were incubated with fluorophore conjugated primary antibody diluted 1:250 and 1:100 in 1X PBS/1% BSA/0.3% Triton™ X-100 overnight at 4°C.
• Cells were rinsed three times in PBS for 5 min each at RT.
• Coverslips were mounted on slides with ProLong® Gold Antifade Reagent with DAPI.
• Isotype control staining:
• All the steps are done same as previously described until cells were blocked in 1X PBS/1% BSA/0.3% Triton™ X-100 to block unspecific binding of the antibodies for 60 min at RT. - Cells were incubated with 10% normal rabbit serum overnight at 4°C.
• Cells were rinsed three times in PBS for 5 min each at RT.
• Cells were incubated with goat anti rabbit CY3 conjugated secondary antibody for 60 min in dark at RT.
• Cells were rinsed three times in PBS for 5 min each at RT.
• Coverslips were mounted on slides with ProLong® Gold Antifade Reagent with DAPI. Secondary only staining:
• All the steps are done same as previously described until cells were blocked in 1X PBS/1% BSA/0.3% Triton™ X-100 overnight at 4°C. - Cells were incubated with goat anti rabbit CY3 conjugated secondary antibody for 60 min in dark at RT.
• Cells were rinsed three times in PBS for 5 min each at RT.
• Coverslips were mounted on slides with ProLong® Gold Antifade Reagent with DAPI.
• Stained cells were imaged with a Nikon C2+ confocal microscope.

Experimental Notes: We did the first set of experiment with 1:250 dilution of the primary antibody but have observed lower level of expression of LRP2 in MCF7 (positive) cells (Figure 2). We repeated experiment with higher concentration of the primary antibody (1:100) and the expression level increased as shown in Figure 1. We have noticed lower level of expression of LRP2 in HeLa (negative) cells (Figure 3). We have used rabbit normal serum for isotype control to match the species with primary antibody (rabbit) and also we used bovine albumin serum (BSA) as blocking and antibody diluent buffer to reduce species cross reactivity.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: Aqueous buffered solution containing 0.01M TBS ( pH 7.4) with 1 % BSA, 0.03 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: -20 °C

Storage Comment: Store at -20°C. Aliquot into multiple vials to avoid repeated freeze-thaw cycles.

Expiry Date: 12 months

Target Details for LRP2

Target: LRP2 (Low Density Lipoprotein Receptor-Related Protein 2 (LRP2))

Alternative Name: Lrp2/Megalin

Background:

Synonyms: DBS, GP33, Low-density lipoprotein receptor-related protein 2, LRP-2, Glycoprotein 33, Megalin, LRP2

Background: Acts together with cubilin to mediate HDL endocytosis (By similarity). May participate in regulation of parathyroid-hormone and para-thyroid-hormone-related protein release.

Gene ID: 4036

UniProt: P98164

Pathways: Metabolism of Steroid Hormones and Vitamin D, Thyroid Hormone Synthesis, Hormone Transport