RIPA Lysis Buffer

Radioimmunoprecipitation assay buffer - most commonly used for cell lysis in western blotting

Components

• 50 mM Tris-HCl, pH 7.4
• 150 mM NaCl
• 1% NP-40 (or IGEPAL CA-630)
• 0.5% Sodium deoxycholate
• 0.1% SDS
• Add fresh before use: 1 mM PMSF, protease inhibitors, phosphatase inhibitors

Preparation

Dissolve components in deionized water, adjust pH to 7.4, filter sterilize, store at 4°C. Add inhibitors just before use. Final volume: 1 L.

Laemmli Sample Buffer (2X)

Standard sample buffer for SDS-PAGE electrophoresis

Components

• 125 mM Tris-HCl, pH 6.8
• 4% SDS
• 20% Glycerol
• 0.004% Bromophenol blue
• 10% 2-mercaptoethanol (add fresh, or use 100 mM DTT)

Preparation

Mix components (except reducing agent), store at room temperature. Add reducing agent just before use. For 2X buffer, mix 1:1 with sample. For reducing conditions, always add fresh 2-ME or DTT.

Wet Transfer Buffer

For transferring proteins from gel to PVDF or nitrocellulose membrane

Components

• 25 mM Tris base
• 192 mM Glycine
• 20% Methanol (for PVDF membranes)
• 0.1% SDS (optional, for large proteins >100 kDa)

Preparation

Dissolve 3.03 g Tris base and 14.4 g glycine in 800 mL deionized water, add 200 mL methanol, adjust pH to 8.3, bring to 1 L. Store at 4°C. For nitrocellulose, reduce methanol to 10-15%.

TBST (Tris-Buffered Saline with Tween-20)

Standard washing and blocking buffer for western blotting

Components

• 20 mM Tris-HCl, pH 7.6
• 150 mM NaCl
• 0.1% Tween-20

Preparation

Dissolve 2.42 g Tris base and 8.77 g NaCl in 900 mL deionized water, adjust pH to 7.6 with HCl, add 1 mL Tween-20, bring to 1 L, mix well. Store at room temperature.

5% Non-Fat Milk in TBST

Standard blocking solution for most western blot applications

Preparation

Dissolve 5 g non-fat dry milk powder in 100 mL TBST. Mix well, may need to filter through cheesecloth to remove undissolved particles. Prepare fresh or store at 4°C for up to 1 week. Shake well before use.

3-5% BSA in TBST

Blocking solution for phosphorylated proteins or when milk causes high background

Preparation

Dissolve 3-5 g BSA (bovine serum albumin, Fraction V) in 100 mL TBST. Mix gently to avoid foaming. Filter through 0.45 μm filter if necessary. Store at 4°C. Use within 1 week.

Ponceau S Staining Solution

For visualizing protein transfer on membrane

Components

• 0.1% Ponceau S
• 5% Acetic acid

Preparation

Dissolve 0.1 g Ponceau S in 100 mL of 5% acetic acid. Filter before use. Store at room temperature. Stains membrane for 2-5 minutes, rinse with water to visualize bands.