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. Apply Fixable Viability Dye in PBS to the cells, as per the manufacturer’s instructions, and mix well. Incubate for 15 min on ice. Typically, 100 μl/well is sufficient. (Optional, but recommended).

2. First layer. Centrifuge the cells and apply V5-tagged Ankyron™ in Wash Buffer, mix well and incubate on ice for 30 mins. We would recommend trying an Ankyron™ titration range from 6 – 0.1 μg per test condition in the first instance.

3. Wash the cells with Wash Buffer and resuspend them in the residual volume.

4. Second layer. Add 8 μl Ankyron™ Anti-V5 Flurotag for Ankyron™ and any additional directly labelled Ankyrons™ or antibodies required for staining to the cells and mix well, incubating samples on ice for 20 – 30 min, shielded from light.

5. Wash the cells twice with Wash Buffer and resuspend thoroughly before adding 200 μl/well 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 Flurotag 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 (0.1 % 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: ELISA Diluent Buffer (1% BSA, 0.05% Tween-20 in PBS), ELISA Wash Buffer (0.1% Tween-20 in PBS).

1. Prepare an ELISA plate as required to the point where an Ankyron™ is required to detect the target of interest. Different Ankyron™ clones may bind different epitopes of the target molecule, making certain clones beneficial for particular target presentation scenarios.

2. Wash wells of the ELISA plate with ELISA Wash Buffer.

3. Add Ankyron™ (V5, His-tagged) to wells of the ELISA plate. We recommend first trying an Ankyron™ titration range from 125ng – 2.5ng / 5mL, where 50μL of the diluted Ankyron™ is added to each well of the ELISA plate.

4. Incubate at room temperature (22°C) for 60 minutes.

5. Wash wells of the ELISA plate with ELISA Wash Buffer.

6. Add Ankyron™ Anti-V5 HRP Tag to wells of the ELISA plate. 50μL of 1:5,000 diluted of Ankyron™ Anti-V5 HRP Tag added to each well of the ELISA plate will be sufficient for most applications.

7. Incubate at room temperature (22°C) for 60 minutes.

8. Wash wells of the ELISA plate with ELISA Wash Buffer.

9. Add 50μL of ECL to each well of the ELISA plate and measure luminescence using a plate reader.


Ankyrons™ in Immunohistochemistry

1. Treat slides either with protease using e.g. the Bond Enzyme Pretreatment Kit (Leica Biosystems cat. no. AR9551) or by heating in e.g. Bond Epitope Retrieval Solution1/2 (Leica Biosystems cat. no. AR9961/AR9649).

2. Block endogenous biotin e.g. with Endogenous Avidin/Biotin Blocking Kit (Zymed, San Francisco, CA, USA).

3. Incubate with biotinylated Ankyron™. We would recommend trying an Ankyron™ concentration titration range between 1 – 5 μg/mL in the first instance.

4. Detect with streptavidin-HRP using 3,3’-diaminobenzidine as a substrate e.g. with the Bond Intense R Detection kit Leica Biosystems, which relies on detection of biotinylated reagents with streptavidin-HRP conjugate, according to the manufacturer’s recommendations.

5. Analyze e.g. with the BondMax staining system (Leica Biosystems).