Western Blot Protocol Checklist
Posted: Mon 8 Dec 2014 17:29Western Blot Protocol Checklist
From Innova Biosciences
Sample Preparation
Research your target protein thoroughly:
- Expression pattern
- Cellular localisation
- Post-translational modification
- Predicted vs. actual size
- Review literature + various websites
Design an effective sample extraction protocol :
- Choose the right buffer – cytoplasmic vs. membrane bound proteins
- Protease inhibitors
- Phosphatase inhibitors
- Fresh buffers
- Clean tools such as homogenisers thoroughly
- Prepare samples quickly and efficiently
- Store appropriately
SDS-PAGE
- Ensure SDS-PAGE rig is clean
- Choose the optimal percentage SDS-PAGE gel
- High vs. low pre-stained molecular weight markers
- Boiling of samples
- Run a reasonable amount of protein - do not overload – important to measure protein concentration
- Balance your lanes to prevent smiling
- Make sure the SDS-PAGE rig is connected to the power supply correctly
- Optimise the running conditions - keeping the system cold, low voltage, overnight vs. 1 hour
Western Blot transfer
- Ensure transfer apparatus is clean
- Wear gloves all the time! Do not contaminate the apparatus
- Wet vs. dry transfer – option depends upon protein
- Membrane – Immobilon® P as preferred choice. Once activated with methanol, do not allow the membrane to dry out.
- Think about using two membranes for smaller proteins
- Equilibrate membrane in transfer buffer for 5 minutes
- Filter paper, membrane + gel – assemble in correct order
- Roll out any trapped bubbles within the system
- Assemble the transfer apparatus following the manufacturer’s protocol
- Connect transfer system to power pack – remember proteins always migrate to the positive - overnight wet transfer - 40V
- Confirm the transfer worked – stain membrane with ponceau S + gel with coomassie blue
Blocking
- Never allow the membrane to dry out – keep hydrated
- Use non-fat dried milk or BSA at 3-5% w/v in TBS or PBS :
• Make sure it is all dissolved
• Store blocking solution in fridge – do not leave at room temperature overnight
- Recommended blocking time 1 hour at room temperature
- Agitate gently using a rocking platform
- Remember to use gloves and tweezers
Primary Antibody
- Optimise antibody dilution – check manufacturer’s guidelines if available
- Buffer optimisation :
• PBS vs. TBS
• Milk vs. BSA
• + or – Tween
• Preferred starting point (TBST + 5% milk)
- Incubation period :
• 1 hour at room temperature or overnight at 4°C
Washing
- Large volume of blocking buffer – 1cm2 = 1ml
- Agitation at room temperature
- 6 x 10 minutes
Secondary Antibody
- Optimise antibody dilution – check manufacturer’s guidelines if available
- Secondary antibody
• HRP conjugated – do not add azide to your buffer
• Fluorescent label – incubate in the dark
Detection
- Remove excess wash buffer using paper towel
- HRP labeled - ECL + film
- Fluorescently labeled – use appropriate machine; keep in dark to avoid bleaching
From Innova Biosciences
Sample Preparation
Research your target protein thoroughly:
- Expression pattern
- Cellular localisation
- Post-translational modification
- Predicted vs. actual size
- Review literature + various websites
Design an effective sample extraction protocol :
- Choose the right buffer – cytoplasmic vs. membrane bound proteins
- Protease inhibitors
- Phosphatase inhibitors
- Fresh buffers
- Clean tools such as homogenisers thoroughly
- Prepare samples quickly and efficiently
- Store appropriately
SDS-PAGE
- Ensure SDS-PAGE rig is clean
- Choose the optimal percentage SDS-PAGE gel
- High vs. low pre-stained molecular weight markers
- Boiling of samples
- Run a reasonable amount of protein - do not overload – important to measure protein concentration
- Balance your lanes to prevent smiling
- Make sure the SDS-PAGE rig is connected to the power supply correctly
- Optimise the running conditions - keeping the system cold, low voltage, overnight vs. 1 hour
Western Blot transfer
- Ensure transfer apparatus is clean
- Wear gloves all the time! Do not contaminate the apparatus
- Wet vs. dry transfer – option depends upon protein
- Membrane – Immobilon® P as preferred choice. Once activated with methanol, do not allow the membrane to dry out.
- Think about using two membranes for smaller proteins
- Equilibrate membrane in transfer buffer for 5 minutes
- Filter paper, membrane + gel – assemble in correct order
- Roll out any trapped bubbles within the system
- Assemble the transfer apparatus following the manufacturer’s protocol
- Connect transfer system to power pack – remember proteins always migrate to the positive - overnight wet transfer - 40V
- Confirm the transfer worked – stain membrane with ponceau S + gel with coomassie blue
Blocking
- Never allow the membrane to dry out – keep hydrated
- Use non-fat dried milk or BSA at 3-5% w/v in TBS or PBS :
• Make sure it is all dissolved
• Store blocking solution in fridge – do not leave at room temperature overnight
- Recommended blocking time 1 hour at room temperature
- Agitate gently using a rocking platform
- Remember to use gloves and tweezers
Primary Antibody
- Optimise antibody dilution – check manufacturer’s guidelines if available
- Buffer optimisation :
• PBS vs. TBS
• Milk vs. BSA
• + or – Tween
• Preferred starting point (TBST + 5% milk)
- Incubation period :
• 1 hour at room temperature or overnight at 4°C
Washing
- Large volume of blocking buffer – 1cm2 = 1ml
- Agitation at room temperature
- 6 x 10 minutes
Secondary Antibody
- Optimise antibody dilution – check manufacturer’s guidelines if available
- Secondary antibody
• HRP conjugated – do not add azide to your buffer
• Fluorescent label – incubate in the dark
Detection
- Remove excess wash buffer using paper towel
- HRP labeled - ECL + film
- Fluorescently labeled – use appropriate machine; keep in dark to avoid bleaching