Ankyron FAQs

Ankyron® Frequently Asked Questions

Do you have recommended protocols for specific Ankyron® applications? Yes! These can be found by heading to the “resources” tab of the navigation bar and selecting “Ankyron® Protocols”. If you have any questions about the specifics of these protocols, please get in touch.

How far does 100µg of Ankyron® go? For flow cytometry 0.1-4 µg can be used to stain 0.3 – 1 × 105 cells depending upon the concentration of Ankyron® found to be optimal from the titration. For Immunofluorescence, 100µg of Ankyron® is sufficient to stain cells in 200 – 400 wells in 96-well plates, depending upon the concentration of Ankyron® found to be optimal from the titration. For IHC, at the recommended dilutions, 100µg can be used to stain ~60 slides

What permeabilization buffer would you recommend for Immunofluorescence microscopy of intracellular and nuclear targets? For intracellular targets we recommend incubation for 30 mins with 0.5% saponin from Quillaja bark. This is a natural product so batch variation means the concentration and incubation time may require optimization. This may also require optimization depending on the specific cell types being stained.

For nuclear targets we recommend incubation for 30 mins with 0.5% TritonX-100. We do not recommend using TritonX-100 for intracellular targets as the permeabilization can be too harsh. Other permeabilization methods e.g. methanol and acetone are not evaluated in house but could be considered when optimizing your protocol.

What permeabilization buffer would you recommend for flow cytometry with intracellular and nuclear targets? We recommend the BD Cytofix/Cytoperm for intracellular targets and the FoxP3/Transcription Factor Fixation/Permeabilization Kit for nuclear targets.

Is my Ankyron® clone suitable for staining fixed cells/ tissue? In house we typically screen Ankyron® clones on both unfixed and formaldehyde fixed cells in addition to fresh/frozen or FFPE tissue. If your clone has associated qualification data with fixed cells/ tissue then your clone is suitable for staining fixed targets. If not, then please contact us for more information, it may be that your clone in unsuitable for staining fixed cells/ tissues or that it has not been tested in these conditions.

What fixation method would you recommend for Immunofluorescence microscopy? We recommend using 1% formaldehydein PBS for 5 minutes, ensuring all formaldehyde has been freshly made (please see here for a recommended recipe). Using higher concentrations and longer incubation times can cause over fixation and prevent target binding. If your application requires higher levels of fixation then please get in touch with us.

What volume of Ankyron® should I use? The concentration (µg/ µL) and amount (µg) of Ankyron® within your aliquot will have provided in your Ankyron® shipment. Please use this information to find the volume required for your protocol.

What is the concentration of my Anti-V5 Fluorotag for Ankyron®? Due to batch variation, we do not detail the precise concentration of each aliquot. This information should not be required as the volume has been optimised to align with the volumes and dilutions stated in our protocols for each technique.

Can I include the pre-conjugation step for Immunofluorescence staining and Immunohistochemistry? Yes! We find that generally this step should not be necessary for these techniques but it can help improve signal for certain targets.

Which Anti-V5 Fluorotag for Ankyron® do you recommend for each technique? For flow cytometry and immunofluorescence, we find the anti-V5 APC usually offers the brightest staining, used with a Cy5/647/APC filter set. For IHC we recommend using the anti-V5 HRP.

Can Ankyrons® be used in Immunoprecipitation? Yes! We recommend using biotin tagged Ankyrons® for this technique. Please get in touch with us to request specific recommendations for using Ankyrons® in IP.

Which wash buffers would you recommend for Immunohistochemistry and Western Blots? For IHC in-house we use the Leica BOND along with the Leica wash buffers. For low throughput IHC and WB, we find the majority of Ankyron® clones perform most effectively with Tris buffers so we would recommend using this initially.

Can I use my Ankyrons® in a sandwich ELISA? Yes! This will require testing pairs of Ankyron® clones to find two that bind distinct epitopes in the correct orientation to find effective sandwich pairs. We testing all available pairs of clones. This requires using a biotinylated clone as the capture Ankyron® and a V5 tagged clone as the detection Ankyron®. Please contact us for a recommended protocol.

Can I use an anti-V5 Fluorotag over an anti-V5 HRP for ELISA? Yes, this is possible. We would recommend a starting concentration of 1 in 1000 but this may require optimization.