Have you tried ultrasonication? What is your protein concentration after your extraction? What is your extraction method for WB? Is it RIPA? Many WB extraction buffer contains detergents that will prevent binding to the plate. One last question, did you confirm that the CDK6 is detectable in WB using the freeze-thaw method or did you just confirm that your cell contains CDK6 using the WB extraction?

Ultrasonication usually keeps the samples pretty clean ( for example no need of SDS that can prevent ELISA from working).

I confirmed the expression of CDK6 using the WB extraction.

The extraction method for WB:

1. Cell Extraction Buffer consists of 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton™ X‑100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate. 2. Immediately before use, add PMSF (0.3 M stock in DMSO) to 1 mM and 50 μL protease inhibitor cocktail for each 1 mL of Cell Extraction Buffer. 3. Collect cells by centrifugation (non‑adherent cells) or scraping from culture flasks (adherent cells), then wash cells twice with cold PBS. 4. Remove and discard the supernatant and collect the cell pellet. The pellet can be stored at −80°C and lysed at a later date if desired. 5. Lyse the cell pellet in Cell Extraction Buffer for 30 minutes, on ice. Vortex at 10‑minute intervals. 6. Transfer the lysate into microcentrifuge tubes and centrifuge at 13,000 rpm for 10 minutes at 4°C. 7. Sonicate the lysate 3 times (10–15 seconds each time). Incubate the lysate on ice between sonication to ensure it stays cold 8. Transfer the supernatant into clean microcentrifuge tubes. Samples can be stored at −80°C (avoid multiple freeze‑thaw cycles).

The protein concentration was 3.821 mg/mL.

With the freeze-thaw method:

The protein concentration was 2.989 mg/mL.

I will use the ultrasonication and see the result.

Thank you

Dear Sir. Concerning your issue about the extraction of nuclear protein for ELISA assay . This protocol has been successfully applied to several cell lines of human origin. Protocol Overview: ELISA Protein Extraction Buffer

1. Prepare 500 µl ELISA protein extraction buffer for each 10-cm dish, or 200 µl for each well in a 6-well dish. Make the solution ice cold and add protease inhibitors, including serine protease inhibitor (such as PMSF). If needed, also add general phosphatase inhibitor.

2. Remove media and rinse cells three times with PBS.

3. Add 500 µl ELISA protein extraction buffer into each 10-cm dish. Rotate the dish to cover cells with ELISA protein extraction buffer and place on a bed of ice for 5 min.

4. Scrape cells off the dish, and transfer to an appropriate tube. Rotate and tap the tube several times to solubilize membranes. You should observe fibrous materials which correspond to genomic DNA, an indicator of cell lysis. If fibrous materials are not apparent, add 20 µl aliquots of ELISA protein extraction buffer, and rotate and tap until the fibrous materials are observed. Keep the tube on ice for another 15 min.

5. Centrifuge the tube at 4oC for 15min and collect the supernatant, which will be used in the ELISA assay. A protein concentration assay, such as the Bradford Assay, can be used to quantify total protein concentration. The supernatant can also be stored at -80oC long term . I think the following below links may help you in your analysis:

https://fivephoton.com/index.php?route=product/product&product_id=190

https://fivephoton.com/pdfs/ELISA%20LYSIS%20AND%20PROTEIN%20EXTRACTION%20BUFFER.pdf

https://www.bosterbio.com/media/pdf/AR0106_DS.pdf

https://www.thermofisher.com/in/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-preparation-protocols/nuclear-extraction-method-.html

Thanks

The cell extraction buffer did not work for the detection of CDK6 by ELISA but worked with WB, maybe because CDK6 is a nuclear protein. So the only option I will use is the cytoplasmic and nuclear extraction buffer.

Thank you for your helpful reply.

Thank you for your reply Kou.