ankyron-protocols

Ankyron™ Protocols

Suggested Ankyron™ Protocols

  1. Ankyrons™ in Flow Cytometry:
    1. Detection of extracellular target proteins
    2. Detection of cytoplasmic target proteins
    3. Detection of nuclear target proteins
  2. Ankyrons™ in Fluorescence Microscopy:
    1. Detection of extracellular target proteins in adherent cells
    2. Detection of intracellular (non-nuclear) target proteins in adherent cells
    3. Detection of nuclear target proteins in adherent cells
  3. Ankyrons™ in ELISA
  4. Ankyrons™ in Immunohistochemistry

*Please note that these protocols are starting point suggestions. They may require optimization for your specific application. We recommend titrating all reagents to determine the optimum quantities required.

Ankyrons™ in Flow Cytometry

Centrifuge Ankyron™ in a chilled microcentrifuge at 14,000 ×g for 5 min. This will remove protein aggregates that contribute to non-specific staining.

Allocate 0.1 – 1 × 106 cells per staining condition, in wells of a 96-well plate. The frequency of the target molecule on the cells of interest will dictate the number of cells required per staining condition and may require optimization.

All steps either on ice or at 4°C, unless otherwise stated, and use ice cold reagents. Low temperature prevents internalization of surface antigens, which could result in reduced fluorescence intensity. Protect all fluorescent reagents from light, to prevent photobleaching.

Detection of extracellular target proteins

Additional materials required: Wash Buffer (0.1% sodium azide, 0.1% BSA in PBS); Fix Solution (1% fetal bovine serum, 2.5% formaldehyde in PBS); Fixable Viability Dye.

1. Centrifuge Ankyron™ Anti-V5 Fluorotag and V5-tagged Ankyron™ in a chilled microcentrifuge at 14,000 ×g for 5 minutes. This will remove protein aggregates that contribute to non-specific staining.

2. Pre-conjugate V5-tagged Ankyron™ in Wash Buffer with anti-V5 Fluorotag for Ankyron™, 8 μl/test. We would recommend trying an Ankyron™ titration range from 4 – 0.1 μg per test condition in the first instance. Allow 50 – 100 μl/sample. Shield from light and incubate at 4 °C for 30 min or overnight.

3. Allocate 0.1 – 1 × 106 cells per staining condition. The frequency of the target molecule on the cells of interest will dictate the number of cells required per staining condition and may require optimization.

4. Wash the cells with an excess volume of Wash Buffer and resuspend them in the residual volume (~50 μl). Keep tubes chilled on ice for all subsequent steps, except where indicated.

5. Add the pre-conjugated V5-tagged Ankyron™ with anti-V5 Fluorotag for Ankyron™ and any additional labelled Ankyrons™ or antibodies required for staining to the cells. Mix well and incubate samples at +4 °C for 30 minutes.

6. Wash the cells twice with an excess volume of Wash Buffer and resuspend thoroughly in the residual volume before adding 200 μl Fix Solution. Store them in Fix Solution in the dark until analysis.

Detection of cytoplasmic target proteins

Additional materials required: Wash Buffer (0.1% sodium azide, 0.1% BSA in PBS); Fix Solution (1% fetal bovine serum, 2.5% formaldehyde in PBS); Fixable Viability Dye; IC Fixation Buffer; Permeabilization Buffer.

1. Apply Fixable Viability Dye in PBS to the cells and mix well. Incubate for 15 min on ice. (Optional, but recommended).

2. If also staining for cell surface proteins, follow steps 2 – 4 in section above.

3. Wash the cells in Wash Buffer, removing as much supernatant as possible.

4. Fixation. Apply IC Fixation Buffer, 200 µl/well, and incubate on ice for 15 min.

5. Dilute Permeabilization Buffer (10x) in 18.2 MΩ water to a working concentration of 1x and chill on ice.

6. Permeabilization. Centrifuge the cells and remove as much supernatant as possible, prior to washing in 1x Permeabilization Buffer, 200 µl/well.

7. First layer. Apply V5-tagged Ankyron™ in 1x Permeabilization Buffer, mix well and incubate for 30 mins. We would recommend trying an Ankyron™ titration range from 6 – 0.1 μg per test condition in the first instance.

8. Wash the cells in 1x Permeabilization Buffer and resuspend them in the residual volume.

9. Second layer. Add 8 μl Ankyron™ Anti-V5 Fluorotag and mix well, incubating samples on ice for 20 – 30 min, shielded from light.

10. Wash the cells twice in 1x Permeabilization Buffer wash buffer and resuspend thoroughly before adding 200 μl Fix Solution. Store them in Fix Solution in the dark until analysis.

Detection of nuclear target proteins

Additional materials required: Wash Buffer (0.1% sodium azide, 0.1% BSA in PBS); Fix Solution (1% fetal bovine serum, 2.5% formaldehyde in PBS); Fixable Viability Dye; FoxP3/Transcription Factor Fixation/Permeabilization Kit. All steps at room temperature, unless otherwise stated. If target protein is localised at cell-cell junctions, the following permeabilization protocol may aid detection.

1. Following steps 1 – 5 in section above, fix samples using the FoxP3 Transcription Factor Staining Kit, as per the manufacturer’s instructions, for 45 min.

2. Permeabilization. Wash fixed samples with FoxP3 Permeabilization Buffer.

3. First layer. Apply His-tagged Ankyron™, 3ug, 150 ul/well in FoxP3 Permeabilization Buffer, mix well and incubate overnight (>12 hours) at room temperature.

4. Wash cells with FoxP3 Permeabilization Buffer 2 – 3 times.

5. Second layer. Apply Ankyron™ anti-His Fluorotag (1/100 dilution) in 100ul FoxP3 Permeabilization Buffer for 1 h at room temperature.

6. Wash cells twice in FoxP3 Permeabilization Buffer, then once in Wash Buffer. before adding 200 μl Fix Solution. Store them in Fix Solution in the dark until analysis.

Ankyrons™ in Fluorescence Microscopy

Centrifuge Ankyron™ in a chilled microcentrifuge at 14,000 xg for 5 min. This will remove protein aggregates that contribute to non-specific staining.

All steps at room temperature, unless otherwise stated.
Do not allow cells to dry out during the staining procedure.
The following protocols are suitable for staining cells in Chambered Cell Culture Slides: polystyrene chambers affixed to glass microscope slides.
After staining, ideally image immediately, or store samples at 4°C in the dark

Detection of extracellular target proteins in adherent cells

Additional materials required:
Wash Buffer (1x Phosphate Buffered Saline, PBS).
Blocking Buffer (10 % v/v Human Serum or Foetal Bovine Serum in RPMI 1640 medium)
If carrying out fixation: Fixative Buffer (4% formaldehyde1 in PBS)

Steps 1 -3 can be omitted if no fixation is required. Simply remove culture media from the cells and proceed with Step 4.

1. Washing. Remove culture media and wash cells with Wash Buffer.

2. Fixation. Apply Fixative Buffer and incubate for 5 min.

3. Washing. Remove Fixative Buffer, apply Wash Buffer then remove, proceeding to Step 4 immediately.

4. First Layer. Apply V5-tagged Ankyron™ in Blocking Buffer, up to 0.08 μg/μl, and incubate for 30 min.

5. Washing. Remove First Layer solution, apply Wash Buffer then remove, proceeding to Step 6 immediately.

6. Second Layer. Apply Ankyron™ Anti-V5 Fluorotag for Ankyron™ in Blocking Buffer, and incubate for 30 min.

7. Washing. Remove Second Layer solution, apply Wash Buffer then remove, proceeding to Step 8 immediately.

8. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g. from BioLegend, use according to the manufacturer’s recommendations.

9. Fixation. Apply Fixative Buffer and incubate for 5 min, then remove Fixative Buffer, applying PBS to the cells in the chambers for the duration of incubation in a solvent bath (typically 10 – 15 min, according to the manufacturer’s instructions) to dissolve the external glue securing the polystyrene chambers to the slide.

10. Mounting. Remove excess fluid and applying mounting medium e.g. VectaShield antifade and coverslip, then apply sealant around the edges, e.g. Biotium CoverGrip Coverslip Sealant

Detection of intracellular (non-nuclear) target proteins in adherent cells

Additional materials required:
Wash Buffer (1x Phosphate Buffered Saline, PBS).
Blocking and Permeabilization Buffer (10 % v/v Human Serum or Foetal Bovine Serum + Saponin (from Quillaja bark, 0.5 % w/v, in RPMI 1640 medium)
If carrying out fixation: Fixative Buffer (4% formaldehyde1 in PBS)

Steps 1 -3 can be omitted if no fixation is required. Simply remove culture media from the cells and proceed to Step 4.
If the target protein is localised in cell-cell junctions, this permeabilization protocol may aid detection.

1. Washing. Remove culture media and wash cells with Wash Buffer.

2. Fixation. Apply Fixative Buffer and incubate for 5 min.

3. Washing. Remove Fixative Buffer, apply Wash Buffer then remove, proceeding to Step 4 immediately.

4. First Layer. Apply V5-tagged Ankyron™ in Permeabilization and Blocking Buffer, up to 0.08 μg/μl, and incubate for 30 min.

5. Washing. Remove First Layer solution, apply Wash Buffer then remove, proceeding to Step 6 immediately.

6. Second layer. Apply Ankyron™ Anti-V5 Fluorotag for Ankyron™ in Permeabilization and Blocking Buffer, and incubate for 30 min.

7. Washing. Remove Second Layer solution, apply Wash Buffer then remove, proceeding to Step 8 immediately.

8. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g., from BioLegend, use according to the manufacturer’s recommendations.

9. Fixation. Apply Fixative Buffer and incubate for 5 min, then remove and apply PBS to the cells in the chambers for the duration of incubation in a solvent bath (typically 10 – 15 min, according to the manufacturer’s instructions) to dissolve the external glue securing the polystyrene chambers to the slide.

10. Mounting. Remove excess fluid and applying mounting medium e.g. VectaShield antifade and coverslip, then apply sealant around the edges, e.g. Biotium CoverGrip Coverslip Sealant

Detection of nuclear target proteins in adherent cells

Additional materials required:
Wash Buffer (1x Phosphate Buffered Saline, PBS).
Nuclear Permeabilization Buffer (0.5 % v/v Triton X-1003 in PBS)
Blocking and Permeabilization Buffer (10 % v/v Human Serum or Foetal Bovine Serum + Saponin (from Quillaja bark, 0.5 % w/v, in RPMI 1640 medium)
If carrying out fixation: Fixative Buffer (4% formaldehyde1 in PBS)

Steps 2 -3 can be omitted if no fixation is required.

1. Washing. Remove culture media and wash cells with Wash Buffer.

2. Fixation. Apply Fixative Buffer and incubate for 5 min.

3. Washing. Remove Fixative Buffer, apply Wash Buffer then remove, proceeding to Step 4 immediately.

4. Nuclear permeabilization. Apply Nuclear Permeabilization Buffer and incubate for 10 min, then wash with PBS.

5. First Layer. Apply V5-tagged Ankyron™ in Permeabilization and Blocking Buffer, up to 0.08 μg/μl, and incubate for 30 min.

6. Washing. Remove First Layer solution, apply Wash Buffer then remove, proceeding to Step 6 immediately.

7. Second layer. Apply Ankyron™ Anti-V5 Fluorotag for Ankyron™ in Permeabilization and Blocking Buffer, and incubate for 30 min.

8. Washing. Remove Second Layer solution, apply Wash Buffer then remove, proceeding to Step 8 immediately.

9. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g., from BioLegend, use according to the manufacturer’s recommendations.

10. Fixation. Apply Fixative Buffer and incubate for 5 min, then remove and apply PBS to the cells in the chambers for the duration of incubation in a solvent bath (typically 10 – 15 min, according to the manufacturer’s instructions) to dissolve the external glue securing the polystyrene chambers to the slide.

11. Mounting. Remove excess fluid and applying mounting medium e.g. VectaShield antifade and coverslip, then apply sealant around the edges, e.g. Biotium CoverGrip Coverslip Sealant

Notes
1. Paraformaldehyde, PFA can be purchased as a methanol-free 16 % w/v aqueous solution in individual ampoules. Dilute to required concentration in PBS and discard 1 week after opening and dilution. Alternatively, PFA can be made in-house from PFA solid, by dissolving in ultrapure water at 2 % w/v, heating to no more than 55°C and adding sodium hydroxide to aid solubility, then adding an equal volume of 2x PBS and adjusting pH to 7.4 using hydrochloric acid (diluted 1 in 10). Again, discard unused PFA after 1 week: over time, formaldehyde in solution will break down into formic acid and methanol, decreasing concentration of formaldehyde available to cross-link and fix tissues. Formic acid may destroy epitopes whilst methanol can disrupt membranes.
2. Saponin, a non-ionic detergent derived from the soapbark tree (Quillaja Saponaria), available from Sigma-Aldrich, to transiently permeabilize the plasma membrane. Once saponin is removed, the membrane will recover and therefore saponin must be included in all steps of the staining protocol
3. Triton X-100, a non-ionic detergent. At high concentrations, it will lyse cells but at this concentration it irreversibly permeabilizes membranes, including the nuclear membrane. The concentration may need to be optimized according to the cell line and may be tested in the range of 0.1 – 0.5 % v/v.
Hazards
Paraformaldehyde/formaldehyde can irritate the skin, throat, lungs and eyes, and is a probable human carcinogen. Use a fume hood if weighing out solid paraformaldehyde. Combustible liquid, harmful if swallowed or in contact with skin, causes serious eye and skin irritation and may be a human carcinogen.
Saponin is an irritant.
Triton X-100 is toxic, irritant and aquatic hazard
Wear protective clothing, safety glasses and gloves when following these protocols.

Ankyrons™ in ELISA

Additional materials required: Diluent Buffer (1% BSA, 0.05% Tween-20 in PBS), Wash Buffer (0.1% Tween-20 in PBS).

1. Add the biotinylated antigen of interest to each well of a streptavidin coated 96-well microtiter plate and incubate for 1 hour at room temperature. Antigens of interest may be bound to a microtiter plate by other methods.
2. Precomplex the Ankyron™ (V5, His-tagged) specific to the antigen of interest with 1x final concentration Anti-V5 HRP Tag for 1 hour at room temperature* (22 °C). We recommend trying an Ankyron™ titration range from 50 – 0.05 ng to each well of the microtiter plate in the first instance. This incubation step to pre-complex the Ankyron™ with antibody increases the signal intensity and thus sensitivity of the assay, as well as reducing experimental time.
3. Wash wells of the microtiter plate with ELISA Wash Buffer.
4. Add the precomplexed Ankyron™ (V5, His-tagged) and Anti-V5 HRP mixture to wells of the microtiter plate.
5. Incubate at room temperature (22 °C) for 1 hour.
6. Wash wells of the microtiter plate with Wash Buffer.
7. Add an HRP compatible substrate to each well of the microtiter plate and measure signal.

*Precomplexing of the bivalent anti-V5 reagent with the V5 tagged Ankyron results in the multimerization of the Ankyron clone increasing the avidity of interaction between Ankyron and target protein.

Ankyrons™ in Immunohistochemistry

Additional materials required

Xylene; 100 % Ethanol; 50 % Ethanol; Wash Buffer (Tris-buffered saline with 0.05 % Tween-20™); Universal Blocker Blocking Buffer (Pierce); anti-V5 HRP; DAB Substrate (e.g. Metal Enhanced DAB Substrate, Pierce); Hematoxylin, Mounting Media; Coverslips
Use ultrapure water (18 MΩ) throughout.

All incubation steps to be carried out at room temperature, unless otherwise stated.

Dewaxing, rehydration and antigen retrieval

Place the slides in a rack and perform the following washes in a solvent-resistant container:

Solution Incubation time
Xylene 5 min
Xylene 5 min
Xylene 5 min
100 % Ethanol 5 min
100 % Ethanol 5 min
50 % Ethanol 5 min
50 % Ethanol 5 min
Water 5 min
Water 5 min

Do not allow sections to dry out, as this will cause non-specific binding of Ankyrons and high background staining.

Transfer slides to Antigen Retrieval Buffer at room temperature and then place in a preheated oven, 60 °C, incubating overnight.

Wash slides in water, prewarmed to 37 °C and proceed immediately to the next step. To prevent wastage of reagents, use a hydrophobic barrier pen to draw a circle around the tissue specimens on the slides.

Ankyron™-mediated detection of target proteins

  1. Quench endogenous peroxidase activity with peroxidase suppressor. This step may be omitted in tissue with low peroxidase present.
  2. Wash slides in Wash Buffer, 3 min x 2.
  3. Apply Universal Blocker Blocking Buffer, 30 min.
  4. Apply V5-tagged Ankyron™, 10 μg/ml in Universal Blocker Blocking Buffer, 30 min.
  5. Wash slides in Wash Buffer, 3 min x 2.
  6. Apply anti-V5 HRP at manufacturer’s recommended concentration, 30 min.
  7. Wash slides in Wash Buffer, 3 min x 2.
  8. Prepare DAB Substrate as per the manufacturer’s instructions, apply to slides and incubate until the desired staining is achieved (typically 5 – 15 min).
  9. Wash slides in Wash Buffer, 3 min x 2.
  10. Rinse with water and drain excess water from the specimen.
  11. Apply Hematoxylin nuclei counterstain to cover the entire tissue surface and incubate for 1 – 2 min.
  12. Drain off the Hematoxylin and wash slides several times with water
  13. Wash slide in Wash Buffer for 1 min
  14. Wash slide in water
  15. Mount slide with Mounting Media and coverslip.