ELISA Protocol
Flow Cytometry Protocol
SDS-PAGE Analysis
SEC-HPLC Analysis
Labeling Antibody with FITC
Labeling Antibody with Biotin
Western Blot Protocol

ELISA Protocol

PRINCIPLE

The enzyme linked immunosorbent assay (ELISA) is a sensible, versatile, precise, reproducible, quantitative and qualitative technique for the determination of antigens or antibodies in a biological sample. ELISA is a plate-based assay technique, which is also named enzyme immunoassay (EIA). The technique was designed for detecting and quantifying substances such as proteins and antibodies. In an ELISA, antigen from the sample is attached to a solid surface and then complexed with an antibody. This antibody is linked to an enzyme or with a labeled secondary antibody, which can catalyze substrates and produces detectable signal, most commonly a color change.

ELISA PROTOCOL

MATERIALS

Reagents

  • Coating Buffer
  • Blocking Buffer
  • Washing Buffer
  • TMB Solution
  • Stopping Buffer
  • Coated Antigen
  • Detect Antibody
  • HRP-conjugated Secondary Antibody

Instruments and Consumables

  • Microplate Reader (CMax Plus)
  • Eppendoff
  • Constant Temperature Incubator

METHODS

1. Coat microtiter plate wells with 100 µl of the antigen at a concentration (e.g. 2 µg/mL) in coating buffer. Incubate overnight at 4℃. Wash the plate 3 times with washing buffer.

2. Add 200 µl of blocking buffer to each well. Incubate for 2 hour at 37℃. Wash 4 times.

3. Add 100 µl of diluted Antibody to the relevant wells. Incubate for 1 hour at 37℃. Wash 4 times.

4. Add 100 µl of HRP-conjugated secondary antibody to each well. Incubate for 1 hour at 37℃. Wash 4 times.

5. Add 100 µl of TMB solution to each well. Incubate at 37℃ for 10-15 minutes.

6. Add 50 µl of stop buffer. Gently tap plate to ensure thorough mixing.

7. Read the OD Value (450 nm).

8. Plate configuration.

Flow Cytometry Protocol

PRINCIPLE

Flow cytometry (FACS or FC) is an immunophenotyping technique that analyze the expression of cell surface and intracellular molecules. Flow cytometry rapidly measures the specific characteristics of a large number of individual cells. Living cells in suspension are stained with specific, fluorescently labeled antibodies and then analyzed with a flow cytometer. The intensity of fluorescence detected by cytometer is directly proportional to the antigen density or the characteristics of the cell being measured.

Flow Cytometry Protocol

MATERIALS

Reagents

  • Washing Buffer
  • Primary Antibody
  • Fluorochrome-labeled
    Secondary Antibody

Instruments and Consumables

  • Test Tube
  • Eppendorf Tube
  • Eppendoff
  • Centrifuge
  • Flow Cytometer

METHODS

1. Harvest, wash the cells and then determine the total cell number.

2. Resuspend the cells to approximately 1-5 x 106 cells/mL in PBS.

3. Add 100 μl of cell suspension to each tube.

4. Add 0.1-10 μg/mL of the primary antibody binding cells antigen. Dilutions, if necessary, should be made in PBS.

5. Incubate for 60 min at 4°C.

6. Wash the cells 3 times by centrifugation at 400g for 5 min and resuspend them in ice cold PBS.

7. Dilute the fluorochrome-labeled secondary antibody in 3% BSA/PBS at the optimal dilution and add to cell suspension.

8. Incubate for 60 min at 4°C in dark.

9. Wash the cells 3 times by centrifugation at 400g for 5 min and resuspend them in PBS.

10. Analyze the cells on the flow cytometer. It is recommended that analysis is performed on the same day.

SDS-PAGE Analysis

PRINCIPLE

SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a biochemical assay to analysis the purity and apparent molecular weight of protein. In this assay, sodium dodecyl sulfate (SDS) is necessary that is an anionic detergent and is used to linearize the proteins and impart a negative charge. The relative migration distance of a protein is negatively proportional to its molecular weight. In order to visually estimate molecular weight, protein maker and coomassie brilliant blue are used in SDS-PAGE. Coomassie brilliant blue is an anionic dye, which binds to proteins and make them visualized as blue bands on a clear background.

SDS-PAGE ANALYSIS

MATERIALS

Reagents

  • 30% Acrylamide
  • 1.5 M Tris-HCl, pH 8.8
  • M Tris-HCl, pH 6.8
  • 10% SDS
  • 10% Ammonium Persulfate
  • TEMED
  • Protein Marker
  • Loading Buffer
  • Running Buffer
  • Coomassie Stain Solution
  • Destain Solution

Instruments and Consumables

  • Electrophoresis Chamber
  • Eppendoff
  • Oscillating Table
  • Incubation Plate

METHODS

Casting the gel

1. Assemble glass plates and spacers in gel casting apparatus.

2. Mix the components for the resolving gel as described above.

3. Pour the resolving gel mixture into the gel plates to a level 2 cm below the top of the shorter plate.

4. Pace a layer of ddH2O over the top of the resolving gel.

5. Allow resolving gel to stand 30 min at room temperature.

6. Drain the ddH2O from top of the resolving gel. Rinse with ddH2O, drain, and wick any remaining ddH2O away with absorbent paper.

7. Mix components for stacking gel.

8. Pour stacking gel solution into top of running gel. Insert comb to the top of the spacers.

9. Allow gel to stand for at least 1 hour at room temperature.

Preparing samples

10. Place a volume of protein solution into a tube.

11. Add a volume of loading buffer.

12. Incubate tubes in boiling water for 10 min.

13. Centrifuge at 12,000g for 30s.

Running the gel

14. Remove comb and assemble cast gel into Electrophoresis chamber.

15. Add freshly prepared running buffer to both chambers of the apparatus.

16. Load the prepared samples into the wells of the gel.

17. Run the gel at 90V until the dye front migrates into the running gel, and increase to 150V until the dye front reaches the bottom of the gel.

Staining and destaining the gel

18. Remove the run gel from the apparatus and remove the spacers and glass plates. Place the gel into an incubation plate.

19. Add staining solution to completely submerge the gel.

20. Stain for 15 min with gentle shaking.

21. Pour off and save the stain.

22. Add destain solution to completely submerge the gel.

23. Destain for 10 min with gentle shaking. Pour off and discard the destain solution.

24. Repeat step 23 until the gel is visibly destained.

25. Pour off and discard the destain solution. Rinse with ddH2O.

26. Record the results.

SEC-HPLC Analysis

PRINCIPLE

Size Exclusion Chromatography (SEC or SEC-HPLC), also known as molecular sieve chromatography, is a technique of purity analysis and macromolecular separation based on molecule size. SEC is a major mode of HPLC that employs porous particles in the column. It is usually applied to determine molecular weight distributions of proteins or separate proteins and polymers used in a wide range of industries.

MATERIALS

Reagents

  • Mobile Phase: 150 mM Phosphate Buffered Saline, pH 7.0

Instruments and Consumables

  • Agilent 1200 Series
  • AdvanceBio SEC 300Å, 7.8 x 300 mm, 2.7 µm, LC column (PL1180-5301, Agilent)

METHODS

Making the Mobile Phase

1. Dissolve Na2HPO4.12H2O and NaH2PO4.2H2O in ultrapure water to make 1000 mL. The resulting solution should have a pH between 6.8 to 7.0.

2. Filter the mobile phase through a 0.22 µm Nylon membrane filter under vacuum to degas the solution and to remove solids that could plug the chromatographic column.

3. Degas the mobile phase via ultrasonication to avoid having a bubble, which could either cause a void in the stationary phase at the inlet of the column or work its way into the detector cell, causing instability with the UV absorbance.

Balance AdvanceBio SEC Column

4. Put line in Mobile Phase solution and unscrew "Purge" valve, set flow rate to 5 mL/min for 3-5 min.

5. Adjusted flow rate to 0.5 mL/min and install chromatographic column, balance for 30-60 min until baseline is stable.

Preparing Sample

6. Adjusted concentration of sample to 1 mg/mL.

7. Filter the sample through a 0.22 µm Nylon membrane filter.

8. Place a volume (~20 µl) of protein solution into sample vials and put on the sample platform of Agilent 1200 Series.

9. Set up system parameters: Wash Location, Inject Location, Inject Volume, Sample Name, Column temperature and Wavelength.

10. Run the sequence.

11. Data analysis.

Labeling Antibody with FITC

PRINCIPLE

Fluorescein isothiocyanate (FITC) is a widely used due to its high quantum efficiency and stability when conjugated. FITC is yellow-orange in color with an absorption maximum at 495 nm. Upon excitation it emits a yellow-green color with an emission maximum at 525 nm. Conjugation occurs through primarily-amine groups of lysine residues, forming a stable thiourea bond. Conjugation of fluorescein isothiocyanate (FITC) to antibody is an extremely valuable technique for identifying surface molecules using either fluorescence microscopy or flow cytometry. The following is the procedure that antibody molecules are coupled with fluorescein derivatives.

MATERIALS

  • 1 to 2 mg/mL purified antibody
  • 5 mg/mL FITC (Prepare the solution fresh)
  • 0.2 M Sodium carbonate-bicarbonate buffer (pH 9.0)
  • Phosphate buffered saline (PBS) buffer
  • Amicon Ultra‐0.5mL (MW= 3,000 Da)
  • Sephadex G-25 column

METHODS

1. Change the antibody solution with 0.2 M Sodium carbonate-bicarbonate buffer (pH 9.0) with G-25 column.

2. Determine antibody concentration based upon A280.

3. Prepare the required dilution of FITC in 0.2 M Carbonate-bicarbonate buffer (FITC labeling buffer).

4. Add FITC labeling buffer to antibody solution and standing for overnight at 4°C.

5. Cover the reaction vial with aluminum foil to protect from light.

6. Separate conjugate from free FITC on G-25 column, and collect fractions.

7. Determine F/P ratio of conjugate spectrophotometrically.

F/P = Moles of FITC / Moles of protein

An F/P of 5 to 6:1 is usually optimal for flow cytometry.

8. Change antibody solution with Phosphate buffered saline (PBS) buffer with G-25 column.

9. Collect the labeled solution. Store at 4°C and protect from light.

Labeling Antibody with Biotin

PRINCIPLE

Biotin labeling assay is primarily used for the preparation of biotin-labeled antibody for enzyme immunoassay (EIA). NH2-Reactive Biotin has a succinimidyl ester group, and can easily make a covalent bond with an amino group of the target protein or other macromolecules.

LABELING ANTIBODY WITH BIOTIN

MATERIALS

Reagents

  • NH2-Reactive Biotin
  • Reaction Buffer
  • WS Buffer

Instruments and Consumables

  • Filtration Tube
  • Incubator
  • Microcentrifuge
  • Microtube

METHODS

1. Add 100 μl WS buffer and the sample solution containing 100 μg protein to a Filtration tube.

2. Pipette to mix and centrifuge at 8,000g for 10 min.

3. Add 10 μl DMSO to NH2-Reactive Biotin, and dissolve with pipetting.

4. Add 100 μL reaction buffer to the filtration tube, and then add 8 μL NH2-reactive biotin solution to the filtration tube and pipette to mix.

5. Incubate the tube at 37°C for 10 min.

6. Add 100 μL WS buffer to the filtration tube, and centrifuge at 8,000g for 10 min. Discard the filtrate.

7. Add 200 μL WS buffer to the filtration tube, and centrifuge at 8,000g for 10 min. Repeat this step one more time.

8. Add 200 μL WS buffer, and pipette about 10 times to recover the conjugate. Transfer the solution to a microtube, and store at 0-5°C.

9. Collect the labeled solution. Store at 4°C and protect from light.

Western Blot Protocol

PRINCIPLE

Western blotting (WB) is a widely practiced analytical technique to detect target proteins using antigen-specific antibodies. The technique involves three major processes: separation by size (Gel electrophoresis), transfer to solid support (Blotting), and marking target protein to visualize (Probing).

Gel electrophoresis is used to separate sample protein based on polypeptide length. The separated protein is transferred onto a nitrocellulose (NC) membrane where it is probed with primary antibody specific to target protein antigen. Typically, the membrane is incubated with antibody against the antigen of interest followed by a secondary antibody and detection.

3-1-7-Western-blot-Protocol

MATERIALS

Reagents

  • Loading Buffer
  • Running Buffer
  • Transfer Buffer
  • Washing Buffer (PBST)
  • Blocking Buffer
  • Chemiluminescent Substrate
  • Protein Maker
  • Primary Antibody
  • Secondary Antibody-HRP

Instruments and Consumables

  • Eppendorf Tube
  • Eppendoff
  • Centrifuge
  • Incubation Plate
  • Oscillating Table
  • Electrophresis Apparatus
  • Transfer Tank
  • Imager

METHODS

Sample preparation

1. Measure the concentration of protein, determine the volume of protein and PBS buffer.

2. Take 16 μL sample and add 4 μL 5x loading buffer. Mix well.

3. Boil the samples for 5 min at 100°C.

4. Centrifuge at 5000 rpm in a microcentrifuge for 1 min.

Protein Separation

5. Load 10 μL sample into the wells of SDS-PAGE gel, along with molecular weight maker.

6. Run the gel at 80 V in stacking gel, about 30 min.

7. Increase the voltage to 100 V in separating gel, about 90 min.

8. Finish the run until the dye front reaches the bottom of the gel.

Protein Transfer

9. Cut NC membrane based on the gel size and wet it in methanol.

10. Assemble the transfer sandwich and make sure no air bubbles between the gel and NC membrane.

11. Place the cassette in the transfer tank, which maintains 4°C in the ice-water bath.

12. Transfer for 60 min at a constant current of 100 V.

Antibody Incubation

13. Block the membrane with 10% skim milk for 60 min at room temperature.

14. Add primary antibody in 5% skim milk and incubate overnight in 4°C on a shaker.

15. Rinse the membrane 3 times with PBST, 8 min at a time.

16. Incubate an HRP-conjugated secondary antibody for 60 min at room temperature.

17. Rinse the membrane 3 times with PBST, 8 min at a time.

Imager and Data Analysis

18. Prepare and apply chemiluminescent substrate (Solution A and B) to the membrane.

19. Capture the chemiluminescent signals using a camera-based imager.

20. Use image analysis software to read the band intensity of the interested proteins.

For research use only, not directly for clinical use.

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