Protease K Antigen Retrieval Buffer
Sample type
FFPE tissues
Assay type
Immunohistochemistry
Concentration
2.0 mg/mL (Stock)
pH (1x)
7.4
Cat no : PR30014
Validation Data Gallery
Product Information
PR30014 is a protease K based solution that can be used for performing protease-induced epitope retrieval (PIER) in immunohistochemistry applications. While treating tissues with formalin and xylene are necessary steps for fixation and deparaffinization in an immunohistochemistry workflow, such treatments often result in protein cross-linking leading to the masking of antigenic sites and the subsequent inhibition of antigen-antibody interactions. Antigen retrieval helps restore the structure of such proteins by breaking the cross-links and unmasking the antigenic sites, thereby making them more accessible to antibodies.
Note: In molecular biology, proteinase K is also known as protease K and is a broad-spectrum serine protease.
Components
0.2 mL Protease K 100x Stock + 20 mL Diluent.
Storage
Shipped at -20°C. Store Protease K 100x Stock at -20°C and Diluent at 4°C or -20°C upon receipt. The product is stable for 6 months from the date of receipt.
Usage
* Thaw the Protease K 100x Stock and the Diluent (if stored at -20°C) before use.
1. Prepare slides from tissues sections following routine methods. Deparaffinize tissues with xylene and re-hydrate using a decreasing ethanol gradient.
2. Wash the slides with washing buffer. Drain off the liquid from the slides and absorb any residual liquid around the tissue. Circle the tissue with a hydrophobic IHC pen (optional).
3. Use Protease K 100x Stockto prepare a 1x working solution by adding the concentrated buffer to the diluent. For example, to make 10 mL of 1x working solution, dilute 0.1 mL Protease K 100x Stock with 9.9 mL Diluent.
4. Apply 100-150 uL of the 1x working solution to the slides and incubate at 37°C for 5-15 minutes in a wet box. Make sure that the retrieval buffer covers the whole tissue. If the tissue is too large, add an additional 50-100 uL of the 1x working solution.
5. Store the remaining undiluted Protease K 100x Stock back at -20°C.
6. Perform subsequent steps of quenching, blocking, primary and secondary antibody incubation, signal development, counter staining and mounting before analyzing the slides.
Application Note
1. Dilute to 1x before use.
2. Calculate how much antigen retrieval buffer will be needed for the experiment to prepare an appropriate amount of 1x working solution. The working solution has a shorter shelf life than the concentrate.