Protein Extraction Solutions (Nuclear, Cytosolic, and Membrane Proteins)

一、Experimental Principle

Protein extraction kits use mild lysis to release total proteins from cells or tissues. Specific binding, centrifugation, and buffer washing remove impurities (e.g., nucleic acids, lipids), yielding pure and active target proteins.

Key Steps:

1. Lysis: Lysis buffer (with detergents, protease inhibitors) disrupts cell membranes to release intracellular proteins.
2. Centrifugation to Remove Debris: High-speed centrifugation removes unlysed cell fragments, nuclei, and large impurities.
3. Protein Purification: Target proteins are selectively adsorbed via centrifugal columns or specific resin, while impurities are discarded with waste liquid.
4. Elution: Low-salt buffer or deionized water elutes target proteins without denaturation.

二、Material Preparation

 Reagent Kit: Protein Extraction Kit (Suitable for various animal, plant cells, tissues, and bacterial samples)

Materials to be Prepared:

- Fresh samples (cells, tissues, etc.)  

- Pre-cooled PBS Buffer （Catalog No：abs962） 

- Centrifuge (capable of reaching 12,000xg)

- Lce bucket or low-temperature operating table

- Vortex mixer （Catalog No：：abs72001）  

- BCA Protein Quantification Kit   （Catalog No：abs9232）

- Liquid nitrogen (for rapid freezing of tissue samples)

三、Experimental Procedure (All lysis steps must be performed on ice or at 4℃)

1、Sample Processing

- Cell Samples: Centrifuge to collect cells (1500xg, 3min), wash twice with PBS, and remove residual liquid.

- Tissue Samples: Cut off 10–50 mg of tissue, quickly freeze in liquid nitrogen, and grind into powder. 

2、Lysis (Refer to the specific instructions of different extraction kits for detailed steps)

- Add 200-500μL of pre-cooled lysis buffer (containing protease inhibitors), and mix by vortexing.

- Lyse on ice for 20-30 minutes, vortexing for 10 seconds every 5 minutes.

3、Centrifugation to Remove Debris

- Centrifuge at 12,000xg at 4℃ for 10 minutes, transfer the supernatant (total protein) to a new centrifuge tube.

-For nuclear proteins, first isolate the nucleus: After hypoosmotic lysis, centrifuge to collect the nuclear pellet, and then dissolve it.  

4、Protein Purification

(Note: Steps 4 and 5 are required for downstream mass spectrometry or enzyme activity analysis)

- Pre-treat the centrifugal column with equilibrium buffer for 5 minutes, centrifuge to discard the waste liquid.

- Add the supernatant of the lysate to the centrifugal column, centrifuge at 12,000xg for 1 minute, and discard the waste liquid.

- Add 500 μL of washing buffer, centrifuge for 1 minute, and repeat twice.

5、Elution of Proteins

- Add 50–100 μL of elution buffer, let it stand for 2 minutes, then centrifuge for 1 minute to collect the eluate (target protein).

6、Protein Storage

- Divide the extracted protein samples into small centrifuge tubes. Store them short-term at -20°C or long-term at -80°C to avoid repeated freeze-thaw cycles.

四、Result Analysis

1、Concentration Determination

   - Determine the protein concentration using the BCA method, and calculate the concentration based on the OD562 reading (using a calibration curve).  

2、Purity Assessment

   - Measure the A260/A280 ratio with a spectrophotometer (ideal value is 1.8–2.0; a value >2.0 suggests nucleic acid contamination).

3、Electrophoresis Verification

   - Assess protein purity via SDS-PAGE electrophoresis: Sharp bands with no tailing or杂质 bands indicate high purity.

五、Common Problems and Solutions

六、Notes

1、Low-temperature operation throughout (on ice or in a 4℃ environment) to prevent protein degradation

2、Prepare lysis buffer freshly before use and avoid repeated freeze-thaw cycles.

3、Tissue samples must be homogenized thoroughly to prevent residual unlysed cells.

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.net

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