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ELISA Protocols

Colorimetric Sandwich ELISA

          The OmniKine™ ELISA Kit contains the components necessary for quantitative determination of natural or recombinant concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a “Sandwich” Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a “sandwich” format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator.

          The capture antibodies coated to the bottom of each well are specific for a particular epitope on while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase.

          After incubation and “sandwiching” of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3’, 5, 5’-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration.

          Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Materials Included:

Component

Quantity Per Kit

Storage/Stability after first use

Microstrips Coated w/ Capture Antibody

12 x 8-Well

Microstrips

1 month at 4°C

Protein Standard

Lyophilized

Biotinylated Detection Antibody

Lyophilized

400x Streptavidin-HRP B

30 μl

Wash Buffer (15x)

50 ml

Assay Diluent 10WR

15 ml

Assay Diluent 1TD

50 ml

Ready-to-Use Substrate

12 ml

Stop Solution

12 ml

Adhesive Plate Sealers

2 Sheets

-

Technical Manual

1 Manual

-

     

Protocol:

1. Prepare all reagents to working concentrations, standards to desired range, and samples to appropriate dilution factors.

2. Remove desired number of capture antibody coated strips for experiment and place remaining strips back into dry pouch with desiccant for 4°C storage.

3. Add 100 μl of Standards/ Samples to each well and incubate on orbital shaker at room temperature (RT) for 2 hrs.

4. Aspirate the solution and add 300 μl of 1x Wash Buffer to each well being       used and gently shake for 2-3 mins on an orbital shaker. Repeat this process 3 times. After the last wash ensure no liquid remains by inverting the plate and tapping it against clean paper towels.

5. Add 100 μl of 1x Detection Antibody to each well and incubate for 2 hrs. on an orbital shaker at RT.

6. Repeat step 4.

7. Add 100 μl of 1x SAV-HRP B to each well and incubate for 30 mins on an orbital shaker at RT

8. Repeat step 4.

9. Add 100 μl of Ready to use Substrate to each well, cover plate from light, and incubate for 20 mins on an orbital shaker at RT.

10. Add 100 μl of Stop Solution to each well and read at 450 nm.

Chemiluminescent Sandwich ELISA

          The LumiAb™ Chemiluminescent ELISA Kit contains the components necessary for quantitative determination of natural or recombinant protein concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a “Sandwich” Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a “sandwich” format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator.

          The capture antibodies coated to the bottom of each well are specific for a particular epitope on proteins while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase.

          After incubation and “sandwiching” of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a sensitive luminescent reaction to ensue upon substrate addition.

          When the Peroxide Enhancer solution is added, the reaction catalyzed by peroxidase yields light that is representative of the antigen concentration. After a brief incubation, the microplate can be read with a luminometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Materials Included:

Component

Quantity Per Plate

Storage/Stability after first use

Microstrips Coated w/ Capture Antibody

12 x 8-Well Microstrips

1 month at 4°C

Protein Standard

Lyophilized

100x Biotinylated Detection Antibody

Lyophilized

400x Streptavidin-HRP

30 μl

Wash Buffer (15x)

50 ml

Assay Diluent 1TD

50 ml

Enhancer Solution

8 ml

Peroxide Solution

8 ml

Adhesive Plate Sealers

2 Sheets

-

Technical Manual

1 Manual

-

Protocol:

  1. Prepare all reagents to working concentrations, standards to desired range, and samples to appropriate dilution factors.

  2. Remove desired number of capture antibody coated strips for experiment and place remaining strips back into dry pouch with desiccant for 4°C storage.

  3. Add 100 µl of Standards/ Samples to each well and incubate on orbital shaker at room temperature (RT) for 2 hrs.

  4. Aspirate the solution and add 300 µl of 1x Wash Buffer to each well being used and gently shake for 2-3 mins on an orbital shaker. Repeat this process 3 times. After the last wash ensure no liquid remains by inverting the plate and tapping it against clean paper towels.

  5. Add 100 µl of 1x Detection Antibody to each well and incubate for 2 hrs. on an orbital shaker at RT

  6. Repeat step 4.

  7. Add 100 µl of 1x SAV-HRP to each well and incubate for 30 mins on an orbital shaker at RT

  8. Repeat step 4.

  9. Add 100 µl of combined Enhancer/Peroxide solution to each well, cover plate from light, and incubate for 5 mins on an orbital shaker at RT.

  10. Read plate on Luminometer.

Colorimetric Cell-Based ELISA

          The Colorimetric Cell-Based ELISA Kit allows for the detection of various target proteins and the effects that certain stimulation conditions have on target protein expression in different cell lines. Qualitative determination of target protein concentration is achieved by an indirect ELISA format.

          In essence, the target protein is captured by target-specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° antibody can catalyze a colorimetric reaction upon substrate addition.

           Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are described: 1) a monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. 2) Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method is used to determine cell density.

          After staining, the results can be analyzed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. 3) If a phosphorylated target is being detected, an antibody against the non- phosphorylated counterpart will be provided for normalization purposes. The absorbance values obtained for the non-phosphorylated target can be used to normalize the absorbance values for the phosphorylated target.

Materials Included:

Reagent

Quantity

Storage/Stability after first use

96-Well Cell Culture Clear-Bottom Microplate

1 Plate

-

10x TBS

24 ml (10x)

1 month at 4°C

Quenching Buffer

24 ml (1x)

Blocking Buffer

50 ml (1x)

15x Wash Buffer

50 ml (15x)

100x Anti-Phospho-GSK3α/β (Tyr279/216) Antibody

60 µl (100x)

100x Anti-GSK3α/β Antibody

60 µl (100x)

100x Anti-GAPDH Antibody(Mouse Monoclonal)

60 µl (100x)

HRP-Conjugated Anti-Rabbit IgG Antibody

12 ml (1x)

HRP-Conjugated Anti-Mouse IgG Antibody

12 ml (1x)

Primary Antibody Diluent

12 ml (1x)

Ready-to-Use Substrate

12 ml (1x)

Stop Solution

12 ml (1x)

Crystal Violet Solution

12 ml (1x)

SDS Solution

24 ml (1x)

Adhesive Plate Seals

2 Seals

-

Protocol:

1) Seed 200 µl of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.

2) Incubate the cells for overnight at 37°C, 5% CO2.

3) Treat the cells as desired.

4) Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.

5) Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. During the incubation, the plates should be sealed with Parafilm. Note: Fixing Solution is volatile. Wear appropriate personal protection equipment (mask, gloves and glasses) when using this chemical.

6) Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. Note: For all wash steps, tap the plate gently on absorbent papers to remove the solution completely.

7) Add 100 µl Quenching Buffer and incubate for 20 minutes at room temperature.

8) Wash the plate 3 times with 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

9) Add 200 µl of Blocking Buffer and incubate for 1 hour at RT.

10) Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

11) Add 50 µl of 1x primary antibodies Anti-Phospho-GSK3α/β (Tyr279/216) Antibody, Anti-GSK3α/β Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature with gentle shaking on the shaker.

12) Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

13) Add 50 µl of 1x secondary antibodies (HRP-Conjugated Anti- Rabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature with gentle shaking on the shaker. Note: Add HRP-Conjugated Anti-Rabbit IgG Antibody to the wells incubated with Anti-Phospho-GSK3α/β (Tyr279/216) Antibody (rabbit, polyclonal) or Anti-GSK3α/β (rabbit, polyclonal) and add HRP-Conjugated Anti-Mouse IgG Antibody to the wells incubated with Anti-GAPDH Antibody (mouse, monoclonal).

14) Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

15) Add 50 µl of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark with gentle shaking on the shaker. Note: Ready-to-Use Substrate is a light-sensitive reagent. Keep away from light.

16) Add 50 µl of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

Optional: Crystal Violet Cell Staining

Crystal Violet binds to cell nuclei and gives absorbance readings proportional to cell counts at 595 nm.

17) After finishing reading the absorbance at 450 nm, wash the plate twice with 200 µl of Wash Buffer and twice with 200 µl of 1x TBS for 5 minutes each. Tap the plates on paper towel to remove the excess liquid. Let plate air dry for 5 minutes at room temperature.

18) Add 50 µl of Crystal Violet Solution to each well, incubate for 30 minutes at room temperature on the shaker. Note: Crystal Violet is an intense stain. Avoid contact with skin and clothing.

19) Tip off Crystal Violet solution into beaker. Wash plate by dipping into bucket of water in the sink with the water continuing to run. Carefully rinse the wells in ddH2O until no more color comes off the wells. Allow the plate to dry for 30 minutes.

20) Add 100 µl of SDS Solution into each well and incubate on the shaker at room temperature for 1 hour.

21) Read absorbance at 595 nm with microplate reader. If absorbance is too high, the solubilized Crystal Violet Solution can be diluted 10 times with ddH2O on a separate 96-well plate.

Fluorometric Cell-Based ELISA

          The Fluorometric Cell-Based ELISA Kit allows for the detection of various target proteins and the effects that certain stimulation conditions have on target protein expression in different cell lines. Qualitative determination of target protein concentration is achieved by an indirect ELISA format.

          In essence, the target protein is captured by target-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of then 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method.

          A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values. If a phosphorylated target is being detected, an antibody against the non-phosphorylated counterpart will be provided for normalization purposes. The RFU values obtained for the non-phosphorylated target can be used to normalize the RFU value for the phosphorylated target.

Materials Included:

Component

Quantity

Storage/Stability after first use

10x TBS

24 ml

1 month at 4°C

Quenching Buffer

24 ml

Blocking Buffer

50 ml

15x Wash Buffer

50 ml

Primary Antibody Diluent

12 ml

100x Anti-BTK (Phospho-Tyr223) Antibody

60 µl

100x Anti-BTK Antibody

60 µl

100x Anti-GAPDH Antibody

110 µl

Dye-1 Conjugated Anti-Rabbit IgG Antibody

6 ml

Dye-2 Conjugated Anti-Mouse IgG Antibody

6 ml

96-Well Black Cell Culture Clear-Bottom Microplate

2 Plates

-

Adhesive Plate Seals

2 Seals

-

Protocol:

1) Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.

2) Incubate the cells for overnight at 37°C, 5% CO2.

3) Treat the cells as desired.

Note: Treatment of cells may result in cell death should be considered prior to treatment.

Note: Vigorous pipetting may knock cells off of the plate. Utilize the well walls to dispense/aspirate gently at each step.

4) Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.

5) Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature.

a.4% formaldehyde is used for adherent cells.

b.8% formaldehyde is used for suspension cells and loosely attached cells.

During the incubation, the plates should be sealed with Parafilm. Note: Fixing Solution is volatile. Wear appropriate personal protection equipment (mask, gloves and glasses) when using this chemical.

6) Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for 3 minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. Note: For all wash steps, tap the plate gently on absorbent papers to remove the solution completely.

7) Add 100 µl Quenching Buffer and incubate for 20 minutes at room temperature.

8) Wash the plate 3 times with 1x Wash Buffer for 3 minutes each time with gentle shaking on the shaker.

9) Dispense 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.

10) Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes at a time with gentle shaking on the shaker.

11) Add 50 µl of “Primary Antibody Mixture P” to corresponding wells for BTK (Phospho-Tyr223) detection. Add 50 ul of “Primary Antibody Mixture NP” to the corresponding wells for total BTK detection. Cover the plate with parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature with gentle shaking.

12) Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time with gentle shaking on the shaker.

13) Add 50 ul of “Secondary Antibody Mixture” to corresponding wells and incubate for 1.5 hours at room temperature with gentle shaking. Note: The plates should be kept in the dark for each step after addition of “Secondary Antibody Mixture”.

14) Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes at a time, with gentle shaking on the shaker. Afterwards, rinse once with 200 ul of 1x TBS. Keep the plate(s) in the dark during wash.

15) Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.

Transcription Factor ELISA

          The TFact DNA-Binding ELISA Kit contains components necessary for detection of active transcription factors in eukaryotic nuclear or cell lysates. This particular immunoassay utilizes the qualitative technique of an indirect ELISA. Streptavidin is bound to the immunoassay plate and specific biotinylated double-stranded (dsDNA) oligonucleotides are then added to bind to the streptavidin via a high affinity biotin-streptavidin interaction.

          After subsequent blocking of extraneous binding sites in each well, the sample containing the target of interest can be added. Primary antibody is added to bind activated transcription factors bound to the dsDNA oligonucleotide, which has been immobilized via the plate-coated streptavidin. A HRP-conjugated secondary antibody specific for rabbit IgGs is added, which allows for specific binding to the Primary Antibody, and consequently colorimetric detection upon addition of the TMB substrate.

          For color development, TMB (3, 3’, 5, 5’-Tetramethylbenzidine) is added to each well. After addition of the substrate, a peroxidase catalyzed reaction will produce a blue TMB Diimine product that is proportional to the target concentration in the sample. Color development is quenched by addition of Stop Solution, or 2 N Sulfuric Acid, which turns the solution yellow. The absorbance can then be read by a spectrophotometer at 450 nm and subsequently allowing for determination of the target concentration in the sample.

Materials Included:

Component

Quality

Container

96-Well dsDNA Oligonucleotide Coated Microplate

12x8-Well Microstrips

-

100x Primary Antibody

100 ul

Yellow

HRP-Conjugated Anti-Rabbit IgG Antibody

12 ml

Brown

Nuclear Lysate Positive Control

Lyophilized

Red

Wild-Type Consensus dsDNA Oligonucleotide

10 ul

Green

Mutant Consensus dsDNA Oligonucleotide

10 ul

Purple

10x Wash Buffer

50 ml

Clear

2x Binding Buffer

12 ml

Clear

Primary Antibody Diluent

12 ml

Clear

Nuclear Wash Buffer

12 ml

Clear

Cytoplasmic Extraction Buffer

6 ml

Brown

Nuclear Extraction Buffer

6 ml

Brown

Ready-to-Use Substrate

12 ml

Brown

Stop Solution

12 ml

Clear

Adhesive Plate Sealers

2 Sheets

-

Technical Manual

1 Manual

-

Protocol:

1. Add 100 μl of Nuclear Lysate Positive Control dilutions or Sample dilutions to the appropriate Primary Antibody or Primary Phospho-Antibody negative control wells. For the negative Nuclear Lysate Positive Control and negative Sample wells, add 100 μl of 1x Binding Buffer.

2. Add 100 μl of WT Oligo Control Dilution into the appropriate WT Oligo Control wells.

3. Add 100 μl of MT Oligo Control Dilution into the appropriate MT Oligo Control wells.

4. Add 100 μl of diluted samples to corresponding wells. For negative sample wells, add 100 μl of 1x Binding Buffer. Incubate plate on orbital shaker at room temperature for 2 hours.

5. Wash 3 times with 1x Wash Buffer with gentle shaking in-between.

6. Add 100 μl of working Primary Antibody or Primary Phospho-Antibody solution to every well that is being. Leave the on orbital shaker at room temperature for 2 hours.

7. Wash 3 times with 1x Wash Buffer with gentle shaking in-between.

8. Add 100 μl of HRP-Conjugated Anti-Rabbit IgG Antibody to each well that is being used. Incubate on orbital shaker at room temperature for 1 hour.

9. Wash 3 times with 1x Wash Buffer with gentle shaking in-between.

10. Add 50 μl of Ready-to-Use Substrate to every well that is being used. Keep those wells away from light and leave on orbital shaker for 10 to 30 minutes until there is distinctive blue color development from the wells. Closely monitor color development as some wells may develop faster than others. The reaction should be terminated when the well with greatest blue color ceases to continue developing.

11. When color development is sufficient, add 100 μl of Stop Solution to each well that is being used. Leave on orbital shaker for 1 minute or shake by hand to ensure color development is completely stopped. There will be a noticeable color change from blue to yellow.

12. The plate is now ready to read. Within 30 minutes of adding Stop Solution, determine the optical density or absorbance of each well by reading on a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from readings at 450 nm.