Proteinase K in Tissue Samples

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• What is the Role of Proteinase K in Isolating Nucleic Acids in Tissue Samples?
• How Do You Isolate Nucleic Acids From Tissue Samples With Proteinase K
• How Do You Isolate Nucleic Acids With Proteinase K From Ffpe Tissue Samples?
• Other Proteinase K Information
• How to Order Large Quantities of Proteinase K
• Materials
• References

What is the role of proteinase K in isolating nucleic acids in tissue samples?

How do you isolate nucleic acids from tissue samples with proteinase K?

The isolation of nucleic acids from tissue samples typically involves several steps, including tissue homogenization, protein digestion with proteinase K, and purification of the nucleic acids. Here is a general overview of the procedure: 

1. Tissue homogenization: The tissue sample is first homogenized to disrupt the tissue matrix and release the nucleic acids. This can be done using a mechanical homogenizer, a blender, or a bead mill.

2. Protein digestion: Proteinase K is added to the homogenized tissue sample to digest the proteins and release the nucleic acids. The amount of proteinase K required can vary depending on the tissue type and the amount of tissue used. Typically, 100-200 μg of proteinase K per mL of tissue homogenate is used. Digestion can be performed at 37-65 °C for 30 minutes to several hours, depending on the tissue type and the extent of protein digestion required. 

3. Inactivation of proteinase K: After protein digestion, the proteinase K is typically inactivated by heating the sample at 95-100 °C for 10-15 minutes. This helps to ensure that the proteinase K is fully inactivated and does not interfere with downstream applications.

4. Nucleic acid purification: The nucleic acids are then purified from the proteinase K-treated sample. This can be done using various methods, including phenol-chloroform extraction, column-based purification, or magnetic bead-based purification. The choice of purification method will depend on factors such as the type and amount of nucleic acid required, the downstream application, and the available equipment.

After purification, the nucleic acids can be quantified using a spectrophotometer or a fluorometer, and their quality can be assessed using gel electrophoresis or other methods. The purified nucleic acids can then be used for downstream applications such as PCR, sequencing, or hybridization assays.

How do you isolate nucleic acids with proteinase K from FFPE tissue samples?

Isolation of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissue samples can be challenging due to the cross-linking of proteins and nucleic acids caused by the formalin fixation process. However, proteinase K can be used to digest the protein cross-links and release the nucleic acids from the tissue. Here is a general overview of the procedure for isolating nucleic acids from FFPE tissue samples with proteinase K:

1. Deparaffinization: The FFPE tissue sample is first deparaffinized to remove the paraffin wax and expose the tissue for subsequent steps. This can be done using xylene or other organic solvents.

2. Rehydration: The deparaffinized tissue is then rehydrated by washing in a series of graded alcohols (e.g., 100% ethanol, 95% ethanol, 70% ethanol, and water).

3. Protein digestion: Proteinase K is added to the rehydrated tissue to digest the proteins and release the nucleic acids. The amount of proteinase K required can vary depending on the tissue type and the amount of tissue used. Typically, 100-200 μg of proteinase K per mL of tissue homogenate is used. Digestion can be performed at 37-65 °C for several hours to overnight, depending on the extent of protein digestion required.

4. Inactivation of proteinase K: After protein digestion, the proteinase K is typically inactivated by heating the sample at 95-100 °C for 10-15 minutes. This helps to ensure that the proteinase K is fully inactivated and does not interfere with downstream applications.

5. Nucleic acid purification: The nucleic acids are then purified from the proteinase K-treated sample. This can be done using various methods, including phenol-chloroform extraction, column-based purification, or magnetic bead-based purification. The choice of purification method will depend on factors such as the type and amount of nucleic acid required, the downstream application, and the available equipment.

After purification, the nucleic acids can be quantified using a spectrophotometer or a fluorometer, and their quality can be assessed using gel electrophoresis or other methods. The purified nucleic acids can then be used for downstream applications such as PCR, sequencing, or hybridization assays.

There are several variations of this protocol, and the specific details can vary depending on the type of nucleic acid being isolated and the downstream application. However, the use of proteinase K digestion is a common step in many protocols for isolating nucleic acids from FFPE tissue samples.

Other Proteinase K Information

To learn how Proteinase K is used blood DNA/RNA extraction, visit How Proteinase K Can Be Used in Blood DNA Extraction.

To learn different Proteinase K protocols here visit Guide on How to Use Proteinase K in Different Procedures.

How do you find the best Proteinase K supplier, visit How to Determine the Best Product and Supplier for Proteinase K.

How to Order Large Quantities of Proteinase K

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