Recombinant Anti-PYK2 antibody [YE353] (ab32571)

Thank you for your kind reminder and your patience. I have been discussing this enquiry with some colleagues.
From what we understand the customer is using this antibody (ab32571: Anti-PYK2 antibody) for cell staining, so I would recommend our ICC protocol - please see the details at the bottom of this message.
The major changes would be to switch to 2% PFA fixation, and omit the antigen retrieval step. They could also modify the blocking step to only 10% serum in PBS for 1 hour (20% serum for cells is very high).
1. Solutions and reagents
1.1. Washing buffer:
PBST washing buffer: 1X PBS / 0.1% Tween-20 (Dulbeccos Phosphate Buffered Saline)
1.2. 2% Paraformaldehyde (prepare fresh by dissolving paraformaldehyde in 1X PBS by heating at 70°C until dissolving. Use cold.)
1.3. 0.1% Triton X-100
1.4. Blocking buffer:
PBS (Dulbeccos Phosphate Buffered Saline) + 10% serum (serum origin depends on the host of the secondary antibody)
1.5. Mounting medium for fluorescence.
1.6. Acetone (Production Grade).
2. Protocol
2.1. Grow cells in chamber slides or in 6-well tissue plates containing sterile coverslips. Prior to fixation, cells should not reach more than 80% confluency.
2.2. Fixation
2.2.1. Remove medium from chamber slides or 6-well plates. Wash once with PBS-T.
2.2.2. Fix cells with 2% paraformaldehyde for 20 min. For HeLa cells only, fix and permeabilize with acetone at -20°C for 5 minutes and continue to step 2.2.5.
2.2.3. Wash cells twice with 1XPBST.
2.2.4. Permeabilize cells with 0.1% Triton-X100 for 5 min
2.2.5. Wash cells twice with 1XPBST.
2.3. Blocking
2.3.1. Block cells with blocking buffer for 1 hour at room temperature (option - O/N 4°C).
2.4. Staining
2.4.1. Dilute primary antibody in the blocking buffer per recommendation on the data sheet.
2.4.2. Apply primary antibody on the cells and incubate overnight at 4°C.
2.4.3. Wash cells twice with 1XPBST.
2.4.4. Incubate cells in a dilution of fluorescently-labeled secondary antibody in PBS for 45 min at room temperature in the dark.
2.4.5. Wash cells three times with 1XPBST.
2.4.6. Counterstain cells with DAPI in a concentration of 300 nM in PBS for 5 min in the dark.
2.4.7. Wash cells three times with 1XPBST.
2.4.8. Mount slides with medium for fluorescent staining.
2.4.9. Store slides in the dark.
I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Thank you for your enquiry regarding ab32571 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.
We have been selling this antibody without any problem so we are not aware of any quality-related issues.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
- Could you please specify the cell types and the species of the samples?
- Were the samples paraffin-embedded sections from tissues?
- If not and the samples are from cultured cells why HIER was applied?
- 20% Serum (normal horse serum) is very high particularly for blocking cells, normally 10% serum or 5% BSA is recommended. If the blocking is too stringent, it can significantly reduce the specific signal.
Generally, PFA fixation does not require antigen retrieval. In fact demasking can destroy the epitope after PFA- fixation. Could you please check with your customer again and confirm if the fixation is 4% PFA or formalin fixed (10% NBF – neutral buffered formalin which is 4% formaldehyde)?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Ja, Sie dürfen die Immunogen-Information gerne in Ihrer Publikation erwähnen.
Ich würde Sie auch gerne bitten die volle Produktnummer (ab32571) anzugeben und uns vielleicht auch eine Kopie Ihrer Publikation zukommen zu lassen. Wir würden uns darüber sehr freuen und würden die Publikation auch auf unser Datenblatt mit aufnehmen.
Ich wünsche Ihnen viel Erfolg mit der Publikation und verbleibe

Vielen Dank für Ihren Anruf.
Es freut mich Ihnen nun die Region, in der das Immunogen liegt, genauer angeben zu können:
Aminosäure 1 bis 30 von humanem PYK2.
Ich kann Ihnen somit auch bestätigen, dass Aminosäure 405 nicht im Immunogen enthalten ist.
Ich hoffe, diese Information ist hilfreich und wünsche Ihnen viel Erfolg bei Ihrer Forschung.