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AlphaLISA AlphaScreen

Preparation of Analyte-depleted Serum for Alpha Assays

Section
Preparation of Analyte-depleted Serum for Alpha Assays
Alpha EMSA Conversion
Alpha Immunogenicity
Alpha Protein-protein and Protein-nucleic Acid Interactions
Alpha Troubleshooting Tables
AlphaLISA Epigenetic Toolbox Reagents
Alpha Instrument Options and Settings
Alpha Bead Conjugation
AlphaLISA 5 μL vs. 20 μL Sample Volume
Alpha Products and Catalog Numbers
Alpha Antibody Detection and Characterization
Alpha Citations
Bead Selection and Bead Interference
The Hook Effect
Other Alpha Applications
AlphaLISA Immunoassay Kits
Preparation of Analyte-depleted Serum for Alpha Assays
Working with cell extracts and supernatants in Alpha assays
Data Analysis for AlphaLISA Immunoassays
Working with Serum and Other Biological Matrices in Alpha Assays
Create your own Alpha Assay
Determining Kd With an Alpha Assay
AlphaLISA and AlphaScreen No-wash Assays
Buffer Selection for Alpha Assays

Overview

The following protocol can be used for serum immunodepletion. You may also be able to purchase analyte-depleted serum from a supplier such as Bioreclamation.

Materials

  • Streptavidin Sepharose: GE Healthcare (catalog number 17-5113-01)
  • Human serum: Cambrex (catalog number 14-402E)

Day 1

Calculations

  • Determine the amount of analyte to be removed from the serum or plasma (based on literature or other assays).
  • Use a 100:1 molar ratio of biotinylated antibody to analyte.
  • Use 20-fold more biotin-binding sites than biotin.

Streptavidin Sepharose preparation

  1. Gently shake the bottle of SA-Sepharose to resuspend the matrix. (Do not vortex!)
  2. Sediment the matrix by centrifugation at 2000 rpm for 5 minutes. Carefully decant the supernatant.
  3. Wash the SA-Sepharose by adding 10 mL of 1X PBS. Invert to mix.
  4. Sediment the matrix by centrifugation at 2000 rpm for 5 minutes.
  5. Repeat washing and centrifugation steps twice more and resuspend in 1X PBS buffer.
  6. Add antibody and incubate for 2 hours at 4ºC (with shaking).

Serum depletion

  1. Sediment the matrix by centrifugation at 2000 rpm for 5 minutes. Carefully decant the supernatant.
  2. Wash the SA-Sepharose by adding 10 mL PBS. (Note: Resuspend the matrix by inverting the tube; do not vortex.)
  3. Sediment the matrix by centrifugation at 2000 rpm for 5 minutes. Carefully decant the supernatant.
  4. Repeat washing and centrifugation steps once more.
  5. Add serum or plasma sample and resuspend the matrix gently.
  6. Incubate 18-24 hours at 4ºC with agitation.

Day 2

Serum recovery

  1. Distribute the SA-Sepharose / serum mix into Eppendorf tubes.
  2. Sediment the matrix by centrifugation at 12,000 rpm for 10 minutes.
  3. Recover as much sample as possible and transfer into new tubes.
  4. Repeat the centrifugation step.
  5. Recover as much sample as possible without aspirating any of the remaining SA-Sepharose and transfer into new tubes.
  6. Sediment any remaining matrix by centrifugation at 13,000 rpm for 10 minutes.
  7. Collect the depleted serum or plasma and store at -20ºC.

Alternatives to Analyte Depleted Serum

Alternatively, a charcoal treatment may be used to remove the analyte of interest. Several protocols are described in the literature:Myrick J.E. et al., (1989), Clin. Chem. 35, pp 37-42 
Glick R.D. et al., (2000), J. Pediat. Surg. 35, pp 465-472 
Raffo A.J. et al., (1995), Cancer Res. 55, pp 4438-4445

An artificial matrix solution could also be prepared. Such a matrix should behave the same way in the AlphaLISA assay as the real samples (same level of signal interference). In such instance, high concentrations of BSA or any other signal quencher could be used for this purpose. Finally, the concentrations of analyte used to perform the calibration curve must cover the range expected to be found in the samples.

 

For research use only. Not for use in diagnostic procedures.

The information provided above is solely for informational and research purposes only. Revvity assumes no liability or responsibility for any injuries, losses, or damages resulting from the use or misuse of the provided information, and Revvity assumes no liability for any outcomes resulting from the use or misuse of any recommendations. The information is provided on an "as is" basis without warranties of any kind. Users are responsible for determining the suitability of any recommendations for the user’s particular research. Any recommendations provided by Revvity should not be considered a substitute for a user’s own professional judgment. 

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