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91899
PathScan® RP ApoE2 Sandwich ELISA Kit
ELISA Kits
ELISA Kit

PathScan® RP ApoE2 Sandwich ELISA Kit #91899

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Figure 1. The PathScan® RP ApoE2 Sandwich ELISA Kit #91899 detects human ApoE2 protein, but not ApoE3 or ApoE4. The absorbance at 450 nm for a panel of cell and tissue extracts (0.05 mg/mL for each sample) using the PathScan® RP ApoE2 Sandwich ELISA Kit #91899 is shown in the left figure. The corresponding western blots using ApoE2 antibody and ApoE (pan) antibody are shown in the right figure (both of these antibodies are human reactive, and not mouse/rat reactive). Extracts were prepared from various cells/tissues, including 293T cells that were either mock transfected (293T/mock) or transfected with constructs expressing full-length human ApoE2 (293T/ApoE2), full-length human ApoE3 (293T/ApoE3), or full-length human ApoE4 (293T/ApoE4).
Figure 2. The relationship between lysate protein concentration from 293T cells transfected with a construct expressing full-length human ApoE2 and the absorbance at 450 nm using the PathScan® RP ApoE2 Sandwich ELISA Kit #91899 is shown.
Figure 3. The PathScan® RP ApoE2 Sandwich ELISA Kit #91899 detects ApoE2 in human cerebrospinal fluid (CSF), plasma, and serum. The absorbance at 450 nm for 3 dilutions of human CSF, plasma, and serum obtained from one Alzheimer's disease (AD) patient and one normal donor is shown in the figure.
To Purchase # 91899
Cat. # Size Qty. Price
91899C
1 Kit  (96 assays)

Important Ordering Details

Custom Ordering Details:

When ordering five or more kits, please contact us for processing time and pricing.

Looking for this ELISA kit in a 384-well format? Inquire for availability, processing time, and pricing.

Supporting Data

REACTIVITY H

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected
Product Includes Volume Solution Color
ApoE (pan) Rabbit mAb Coated Microwells 96 tests
ApoE2 Rabbit Detection mAb 1 ea Red (Lyophilized)
HRP Diluent 5.5 ml Red
TMB Substrate 7004 11 ml
STOP Solution 7002 11 ml
Sealing Tape 2 ea
ELISA Wash Buffer (20X) 9801 25 ml
Cell Lysis Buffer (10X) 9803 15 ml

Product Description

The rapid protocol (RP) PathScan® RP ApoE2 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of ApoE2 in a reduced assay time of 1.5 hours. Incubation of cell lysates and detection antibody on the coated microwell plate forms a sandwich with ApoE2 in a single step. The plate is then extensively washed and TMB reagent is added for signal development. The magnitude of absorbance for the developed color is proportional to the quantity of ApoE2. Learn more about all of your ELISA kit options here.

*Antibodies in this kit are custom formulations specific to kit.

Protocol

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PathScan® Sandwich ELISA Protocol (Rapid Protocol)

NOTE: This protocol is for PathScan® kits that use an HRP directly conjugated to the detection antibody (Rapid Protocol), rather than a 2-step method where the detection antibody and a secondary-HRP are added sequentially.

A. Solutions and Reagents

NOTE: Prepare solutions with deionized/purified water or equivalent.

  1. Microwell strips: Bring all to room temperature before opening bag/use. Unused microwell strips should be returned to the original re-sealable bag containing the desiccant pack and stored at 4°C.
  2. Detection Antibody: Reconstitute lyophilized Detection Antibody (red colored cake) with 5.5 mL HRP Diluent. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. For best results, use immediately following antibody reconstitution. Unused reconstituted Detection Antibody may be stored for up to 4 weeks at 4°C, although there may be some loss of signal compared to freshly reconstituted antibody.
  3. HRP Diluent: Red colored diluent for reconstitution and dilution of the Detection Antibody that is linked to HRP.
  4. 1X ELISA Wash Buffer: Prepare by diluting ELISA Wash Buffer (20X) (included in each kit) to 1X with deionized water.
  5. 1X Cell Lysis Buffer: Prepare by diluting 10X Cell Lysis Buffer #9803 to 1X with deionized water. This buffer can be stored at 4°C for short-term use (1–2 weeks). Recommended: When using to prepare cell lysates, add Protease/Phosphatase Inhibitor Cocktail (#5872, not supplied) and 1 mM phenylmethyl- sulfonyl fluoride (PMSF, #8553, not supplied) immediately before use.
  6. TMB Substrate (#7004): Bring to room temperature before use.
  7. STOP Solution (#7002): Bring to room temperature before use.

B. Preparing Cell Lysates

For adherent cells

  1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 mL ice-cold 1X Cell Lysis Buffer including 1 mM PMSF and Protease/Phosphatase Inhibitor Cocktail to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
  4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  5. Sonicate lysates on ice.
  6. Microcentrifuge for 10 min (14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/mL. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1200 rpm) and wash once with 5-10 mL ice-cold 1X PBS.
  3. Cells harvested from 50 mL of growth media can be lysed in 2.0 mL of 1X Cell Lysis Buffer including 1 mM PMSF and Protease/Phosphatase Inhibitor Cocktail.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at −80°C in single-use aliquots.

C. Test Procedure

NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.

  1. Prepare all reagents as indicated above (Section A).
  2. Samples should be undiluted or diluted with 1X Cell Lysis Buffer to a 2X protein concentration in order to achieve a final 1X protein concentration upon addition of the Detection Antibody. Individual datasheets for each kit provide a sensitivity curve that serves as a reference for selection of an appropriate starting lysate concentration. The sensitivity curve shows typical results across a range of lysate concentration points.
  3. Add 50 µL of each sample to the appropriate wells.
  4. Add 50 µL of the Detection Antibody to each well.
  5. Seal the plate and incubate for 1 hour at room temperature on a plate shaker set to 400 rpm (moderate agitation).
  6. Gently remove the tape and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash 4 times with 1X Wash Buffer, 200 µL each time for each well.
    3. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
    4. Clean the underside of all wells with a lint-free tissue.
  7. Add 100 µL of TMB Substrate to each well. Seal with tape and incubate the plate in the dark for 15 min at room temperature on a plate shaker (400 rpm, moderate agitation) or alternatively for 10 min at 37°C without shaking.
  8. Add 100 µL of STOP Solution to each well. Shake gently for a few seconds.
  9. NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

  10. Read results:
    1. Visual Determination: Read within 30 min after adding STOP Solution.
    2. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP Solution.

created July 2020

Protocol Id: 2144

Specificity / Sensitivity

The PathScan® RP ApoE2 Sandwich ELISA Kit detects endogenous levels of ApoE2 protein, but does not cross-react with ApoE3 or ApoE4, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Background

Apolipoproteins are plasma lipoproteins that function as transporters of lipids and cholesterol in the circulatory system. Chylomicrons are a fundamental class of apolipoproteins containing very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) (1,2).
Human ApoE has three isoforms: ApoE2, ApoE3, and ApoE4. These three isoforms differ in the combination of cysteine and arginine residues located at positions 130 and 176. The ApoE2 isoform contains cysteine at both locations. ApoE4 has been identified as a genetic risk factor for Alzheimer’s disease (AD), where ApoE2 is neuroprotective, reducing risk of AD by nearly 50%. The neuroprotective mechanism of ApoE2 remains under investigation, but studies suggest that this may be due to its overall protein abundance in non-AD human cortex and its unique lipid binding profile compared to ApoE3 and ApoE4 which may increase overall beta-amyloid clearance (3).

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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