Sample Preparation from Tissues

Tissue homogenization and extraction for western blot analysis using denaturing or mild lysis buffers.

This protocol page covers three tissue preparation methods: a general Lysate Sample Preparation for extracting total soluble proteins, a Brain Synaptosomes Sample Preparation for isolating synapse-enriched brain fractions, and an Enriched Membrane Fraction Preparation for selectively pelleting membrane components. Together, they provide versatile approaches for preparing high-quality tissue samples for Western blot. For cell line samples, refer to our Sample Preparation from Cell Lines Protocol.

Procedure

Step 1 - Tissue Collection and Storage

• Remove the tissue of interest from the animal.
• Flash-freeze immediately in liquid nitrogen.
• Store at –80°C until use.
  
  

Step 2 - Initial Homogenization

• Homogenize frozen tissue in 5 volumes of the appropriate buffer using a motorized high-speed homogenizer:
    a. Enriched Membrane Fraction Preparation → use Lysis Buffer A
    b. Brain Synaptosomes (P2 protocol) → use Lysis Buffer B
    c. Tissue Lysate Preparation → use Lysis Buffer C

Method A — Enriched Membrane Fraction Preparation

Step 3a - First Low-Speed Centrifugation

• Centrifuge the homogenate in a pre-cooled centrifugation
  ⏱ 2,000 × g for 10 min at 4°C.
• Transfer the supernatant to a clean cold tube, leave on ice, and continue with pellet.
  
  

Step 4a - Re-Homogenization of Pellet

• Resuspend the pellet in 2 volumes of ice-cold Lysis Buffer A.
• Homogenize with manual homogenizer briefly on ice.
• Centrifuge in a pre-cooled centrifugation
  ⏱ 2,000 × g for 10 min at 4°C.
• Combine this supernatant with the supernatant collected in Step 3.
  
  

Step 5a - High-Speed Membrane Isolation

• Centrifuge the extraction using pre-cooled ultracentrifugation
  ⏱ 100,000 × g for 1 hour at 4°C.
• Discard the supernatant.
  The pellet contains the enriched membrane fraction.
  
  

Step 6a - Membrane Pellet Resuspension

• Resuspend the pellet in 2 volumes of cold Lysis Buffer A.
• Homogenize briefly with mechanical homogenizer, on ice.
  
  

Step 7a - Protein Quantification and Storage

• Determine protein concentration conducting Bradford assay.
• Adjust to 4 mg/ml with Lysis Buffer A before storing at –80°C until further use.

Method B — Brain Synaptosomes (P2 Fraction)

Step 3b - Low-Speed Clarification

• Centrifuge the homogenate in a pre-cooled centrefugation
  ⏱ 700 × g for 10 min at 4°C.
• Transfer the supernatant to a clean pre-cooled tube (discard pellet).
  
  

Step 4b - P2 Fraction Isolation

• Centrifuge the supernatant
  ⏱ 37,000 × g for 40 min at 4°C.
• Discard the supernatant. The pellet contains the P2 synaptosomal fraction.
  
  

Step 5b - Extraction of Synaptosomal Proteins

• Resuspend the P2 pellet in half the original homogenization volume using Extraction Buffer.
• Incubate
  ⏱ 37°C for 30 min.
  
  

Step 6b - High-Speed Clarification

• Centrifuge
  ⏱ 100,000 × g for 60 min at 4°C.
• Transfer the clear supernatant (enriched synaptosomal extract) to a clean tube.
  
  

Step 7b - Protein Quantification and Storage

• Determine protein concentration (Bradford assay). 
• Adjust to 4 mg/ml with Extraction Buffer.
• Store at –80°C until further use.

Method C — Tissue Lysate Preparation

Step 3c - Extraction

• Incubate the lysate on a rotator
  ⏱ 30 min at 4°C.
  
  

Step 4c - Clarification

• Centrifuge the lysate
  ⏱ 100,000 × g for 1 hour at 4°C.
• Transfer the supernatant (total tissue lysate) to a clean pre-cooled tube.
  
  

Step 5c - Protein Quantification and Storage

• Determine protein concentration with Bradford assay.
• Adjust to 4 mg/ml with Lysis Buffer C.
• Store at –80°C until further use.

Troubleshooting and Tips