Sample Preparation Techniques

Sample Collection and Storage

 Types of Biological Samples:

• Tissue samples: Obtained from various organs or biopsies.

• Cell lysates: Derived from cultured cells or isolated from specific tissues.
• Body fluids: Such as blood plasma, urine, cerebrospinal fluid, and saliva.

 Proper Storage Conditions:

• Immediate freezing in liquid nitrogen and storage at -80°C to prevent protein degradation.
• Use of protease inhibitors to prevent proteolytic cleavage during storage.
Protein Extraction and Solubilization

 Methods for Protein Extraction:

• Mechanical disruption: Homogenization, sonication, or bead beating to break cell walls and release proteins.
• Chemical lysis: Use of lysis buffers containing detergents and chaotropes to solubilize proteins.

 Use of Detergents, Chaotropes, and Reducing Agents:

•Detergents: SDS, Triton X-100, and NP-40 to solubilize membrane proteins and maintain them in solution.
•Chaotropes: Urea and thiourea to disrupt hydrogen bonds and denature proteins, enhancing solubility.
•Reducing agents: Dithiothreitol (DTT) or β-mercaptoethanol to break disulfide bonds, ensuring proteins are in
their reduced form.
Protein Quantification and Quality Control
 Techniques for Quantifying Protein Concentration:

• Bradford Assay: A colorimetric assay based on the binding of Coomassie Brilliant Blue
dye to proteins, causing a shift in the dye's absorption maximum.

• BCA Assay (Bicinchoninic Acid Assay): A colorimetric assay where proteins reduce Cu²⁺
to Cu⁺ under alkaline conditions, which then forms a purple complex with bicinchoninic
acid.
 Assessing Sample Quality and Purity:
• SDS-PAGE: Running a small aliquot on a gel to check the protein pattern and ensure no
degradation or contamination.
• Spectrophotometric Analysis: Measuring absorbance at 280 nm to estimate protein
concentration and at 260 nm to check for nucleic acid contamination.

(Spectrophotometric analysis of the GST removal)

 Detailed Steps in Sample Preparation

1. Collection:
•Collect samples quickly to prevent degradation.
•Use appropriate methods to minimize protease activity (e.g., using protease inhibitors).

2. Storage:
•Flash freeze samples in liquid nitrogen and store at -80°C until further processing.

3. Lysis and Extraction:

• Mechanical Disruption: If dealing with tissue or cell pellets, use homogenizers or sonicators
to physically break cells.
• Chemical Lysis: Add lysis buffer containing detergents, chaotropes, and reducing agents to
solubilize proteins.
• Incubation: Allow the sample to incubate on ice or at room temperature for a specific
duration to ensure complete lysis.
4. Centrifugation:
• Centrifuge the lysate at high speed to remove cell debris and insoluble material.
• Carefully collect the supernatant containing soluble proteins.

5. Quantification and Quality Check:

• Perform a protein assay (Bradford or BCA) to determine the protein concentration.
• Run a small aliquot on an SDS-PAGE gel to check for integrity and purity of the proteins.