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The procedure below provides a general guidance for the preparation of commonly tested samples for use in ELISA assays. Please check with the literature for experiments similar to yours for your new assay development. In this article, the following samples are covered:

  1. Cell Culture Supernatant
  2. Cell Culture (Conditioned) Media
  3. Cell Lysate
  4. Tissue Homogenate
  5. Tissue (Bone)
  6. Serum
  7. Plasma
  8. Urine
  9. Saliva
  10. Milk
  11. General tips
  1. Cell Culture Supernatant

    • Centrifuge cell culture media at 1,500 rpm and 4°C for 10 min.
    • Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).
  2. Cell Culture (Conditioned) Media

    Since serum tends to contain cytokines which may produce significant background signals, we suggest the preparation of serum-free or low-serum medium samples. If it is necessary to test serum containing medium, we also suggest running an uncultured medium blank to obtain the baseline signals which can then be subtracted from the cultured media sample data.

    Day 1

    • Plate cells in complete growth media (with serum) until the desired level of confluence is achieved.
    • Note: The optimal number of seeded cells varies from one cell type to another and may need to be empirically determined. We suggest seeding around 1 million cells in 100 mm tissue culture plate with complete growth medium. 

    Day 4

    • Remove growth media and gently wash cells using 2-3 mL of warm PBS.
    • Repeat the wash step.
    • Remove PBS and replace the medium with serum-free or low serum containing medium (e.g. medium containing 0.2% calf serum).  
    • Incubate for 1-2 days.

    Day 6

    • Collect medium.
    • Centrifuge at 1,500 rpm and 4°C for 10 min.
    • Aliquot the supernatant and keep it at -80ºC until experiment (Avoid freeze/thaw cycles). Most samples prepared this way can be stored for at least one year. 
  3. Cell Lysate

    • Collect and rinse cells in PBS.
    • Homogenize and lyse cells thoroughly in lysis buffer (e.g. Mammal Cell Protein Extraction Reagent, Boster Catalog Number: AR0103).
    • Centrifuge cell lysate at approximately 10,000 x g and 4°C for 5 min.
    • Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).

    Guidelines on lysis buffer

    • Avoid more than 0.1% SDS or other strongly denaturing detergents (In general, non-ionic detergents such as Triton X-100 or NP-40 are best. Zwitterionic detergents such as CHAPS or mild ionic detergents such as sodium deoxycholate will work). 
    • Do not use more than 2% v/v total detergent.  
    • Avoid the use of sodium azide. 
    • Avoid more than 10 mM reducing agents (e.g. dithiothreitol, mercaptoethanols). 
  4. Tissue Homogenate

    • Rinse tissue with PBS to remove excess blood.
    • Chop tissue into 1-2 mm pieces on ice in ice-cold buffer, preferably in the presence of protease inhibitors.
    • Place the tissue in micro-centrifuge tubes and dip them into liquid nitrogen to snap freeze.
    • Keep samples at -80°C for later use or keep on ice for immediate homogenization.
    • Prepare the extraction buffer. It can be prepared ahead of time and stored at 4°C.
      • 100 mM Tris, pH 7.4
      • 150 mM NaCl
      • 1 mM EGTA
      • 1 mM EDTA
      • 1% Triton X-100 0.5%
      • 0.5% sodium deoxycholate
    • Immediately before use, the extraction buffer must be supplemented with the following to generate a complete extraction buffer.
      • Phosphatase inhibitor cocktail [as directed by manufacturer]
      • Protease inhibitor cocktail [as directed by manufacturer]
      • PMSF (Phenyl Methyl Sulfonyl Floride) to 1 mM
    • For every 5 mg of tissue, add 300 µL of complete extraction buffer to the tube and homogenize.
    • Rinse the blade of the homogenizer 2X with 300 µL extraction buffer.
    • Place the sample on a shaker at 4°C for 2 hours.
    • Centrifuge the sample at 13,000 rpm and 4°C for 20 min.
    • Assay immediately or aliquot supernatant (soluble protein extract) and hold at -80°C (Avoid freeze/thaw cycles).
  5. Tissue (Bone)

    • Extract de-mineralized bone samples in 4M Guanidine-HCl and protease inhibitors.
    • Dissolve the final sample in 2M Guanidine-HCl.
  6. Serum

    • Collect whole blood into a tube without additives.
    • Keep the blood at room temperature for 30 min.
    • Centrifuge at 3,000 rpm and 4°C for 10 min.
    • Assay immediately or aliquot supernatant (serum) and hold at -80°C (Avoid freeze/thaw cycles).

    Note: Hemolysis should be avoided while collecting serum samples. Those samples that have undergone hemolysis may increase non-specifically staining in HRP-conjugated ELISA assay.

  7. Plasma

    • Collect whole blood into a tube containing anti-coagulant (Different protein may require different anti-coagulant, see datasheet for details on what anti-coagulant use).
    • Centrifuge at 3,000 rpm and 4°C for 10 min.
    • Assay immediately or aliquot supernatant (plasma) and hold at -80°C (Avoid freeze/thaw cycles).
  8. Urine

    • Collect urine into sterile or disposable container (Fresh urine sample must be used immediately or saved to avoid reproduction of bacteria which produce endogenous HRP for giving potential false positive results).
    • Centrifuge sample at 10,000 x g for 1 min.
    • Assay immediately or aliquot supernatant and hold at -80°C. (Avoid freeze/thaw cycles).
  9. Saliva

    • Collect saliva using collection device without any protein binding or filtering capabilities (e.g. Salivette).
    • Centrifuge at 10,000 x g and 4°C for 2 min.
    • Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).
  10. Milk

    • Centrifuge at 1500 x g and 4°C for 15 min.
    • Collect the aqueous fraction and repeat 3X this process.
    • Filter through a 0.2 µm filter.
    • Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).

General tips for ELISA sample preparation:

  • Serum, plasma, cell and tissue extracts are typically diluted by 50% with binding buffer.
  • Total protein concentration of homogenate should be at least 1 mg/mL. However, 2 mg/mL or more would be better.
  • The collected samples can be kept for different periods: 48 hours (2-8°C), 1 month (-20°C) or 6 months (-70°C).
  • The protein concentration of your lysates can be determined by a total protein assay not inhibited by detergents such as the Bicinchoninic acid (BCA) assay. The volume of each sample can also be normalized to deliver the same amount of total protein for each assay.