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.
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.
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.
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.
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.
Additional materials required: Wash Buffer (1x Phosphate Buffered Saline, PBS); Fixative buffer (1% paraformaldehyde1, see notes in PBS); Blocking Buffer (1% w/v Bovine Serum Albumin + 5 % v/v Foetal Bovine Serum).
All steps at room temperature, unless otherwise stated.
1. Washing. Remove media and wash cells with Wash Buffer.
2. Blocking. Apply Blocking Buffer and incubate for 20 min.
3. First layer. Apply V5-tagged Ankyron™ in Blocking Buffer, 1:500, and incubate for 45 min.
4. Washing, 3 x 5 mins in Wash Buffer.
5. Second layer. Apply Ankyron™ Anti-V5 Flurotag for Ankyron™ in Blocking Buffer, and incubate for 45 min.
6. Washing, 3 x 5 mins in Wash Buffer.
7. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole), use according to the manufacturer’s recommendations.
8. Fixation. Apply Fixative buffer and incubate for 5 min, then wash briefly with Wash Buffer.
9. Mounting. Applying mounting medium e.g., VectaShield antifade and coverslip, then apply sealant around the edges, e.g., Biotium CoverGrip Coverslip Sealant.
Additional materials required: Wash Buffer (1x Phosphate Buffered Saline, PBS); Fixative buffer (1% paraformaldehyde in PBS); Permeabilization and Blocking Buffer (0.2% w/v Saponin), 1% w/v Bovine Serum Albumin + 5 % v/v Foetal Bovine Serum in 1x Phosphate Buffered Saline).
All steps at room temperature, unless otherwise stated. If target protein is localized at cell-cell junctions, the following permeabilization protocol may aid detection.
2. Fixation. Apply Fixative buffer and incubate for 10 min.
3. Washing, 3 x 5 mins in Wash Buffer.
4. Blocking and permeabilization. Apply Permeabilization and Blocking Buffer and incubate for 20 min.
5. First layer. Apply V5-tagged Ankyron™ in Permeabilization and Blocking Buffer, 1:500, and incubate for 45 min.
6. Washing, 3 x 5 min in Wash Buffer.
7. Second layer. Apply Ankyron™ Anti-V5 Flurotag for Ankyron™ in Permeabilization and Blocking Buffer, and incubate for 45 min.
8. Washing, 3 x 5 mins in Wash Buffer.
9. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g., from Invitrogen, use according to the manufacturer’s recommendations.
10. Fixation. Apply Fixative buffer and incubate for 5 min, then wash briefly with Wash Buffer.
11. Mounting. Applying mounting medium e.g., VectaShield antifade and coverslip, then apply sealant around the edges, e.g., Biotium CoverGrip Coverslip Sealant
Additional materials required: Wash Buffer (1x Phosphate Buffered Saline, PBS); Fixative buffer (1% paraformaldehyde in PBS); Permeabilization Buffer (0.3 % v/v Triton X-1003 in PBS); Permeabilization and Blocking Buffer (0.2% w/v Saponin), 1% w/v Bovine Serum Albumin + 5 % v/v Foetal Bovine Serum in 1x Phosphate Buffered Saline). All steps at room temperature, unless otherwise stated.
3. Washing, 3 x 5 min in Wash Buffer.
4. Permeabilization. Apply Permeabilization Buffer, 10 min.
5. Washing, 3 x 5 mins in Wash Buffer.
6. Blocking and permeabilization. Apply Permeabilization and Blocking Buffer and incubate for 20 min.
7. First layer. Apply V5-tagged Ankyron™ in Permeabilization and Blocking Buffer, 1:500, and incubate overnight at 4°C.
8. Washing, 3 x 5 min in Wash Buffer.
9. Second layer. Apply Ankyron™ Anti-V5 Flurotag for Ankyron™ in Permeabilization and Blocking Buffer, and incubate for 45 min.
10. Washing, 3 x 5 mins in Wash Buffer.
11. Nuclei Counterstain. DAPI (4′,6-Diamidino-2-Phenylindole) e.g., from Invitrogen, use according to the manufacturer’s recommendations.
12. Fixation. Apply Fixative buffer and incubate for 5 min, then wash briefly with Wash Buffer.
13. Mounting. 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 the 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 the concentration suggested here, 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.
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.
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).
