This protocol has more steps and requires more reagents than a conventional Western blot, but it greatly enhances signal from proteins with low abundance. For more information see “Improving the Sensitivity of Traditional Western blotting via Streptavidin containing Poly Horseradish Peroxidase (PolyHRP)” Manish Mishra et al.

Equipment

Mini Gel Tank (ThermoFisher, A25977)	Oscillating platform in refrigerator
Gel spatula	Glass trays
Trans-Blot Turbo Transfer System (Bio-Rad)	Plastic lidded boxes
Gel roller	Incubator
Plastic forceps	Tube rotator
Rocking platform	Digital imager

Materials

• Protein sample
• 4X Bolt™ LDS Sample Buffer (ThermoFisher, B0008)
• 10X Bolt™ Sample Reducing Agent (ThermoFisher, B0009)
• PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (ThermoFisher, 26616)
• Bolt™ Bis-Tris Plus Mini Protein Gels, 4-12% (ThermoFisher, NW04122BOX)
• 20X Bolt™ MES SDS Running Buffer (ThermoFisher, B0002)
• Bolt™ Antioxidant (ThermoFisher, BT0005)
• No-Stain™ Protein Labeling Reagent (ThermoFisher, A44717)
• Trans-Blot Turbo RTA Mini 0.45 µm LF PVDF Transfer Kit (Bio-Rad, #1704274)
• Acetone
• Methanol
• 20X Tris-buffered saline with tween-20 (TBS-T) (ThermoFisher, 28360)
• Non-fat dry milk (Cell Signaling Technology, #9999)
• Molecular Probes™ D-Biotin (ThermoFisher, B1595)
• Streptavidin (MedChem Express, HY-P3152-10mg)
• Bovine serum albumen (Cell Signaling Technology, #9998)
• Primary antibodies
• Biotinylated secondary antibodies (e.g. Goat anti Rabbit IgG (H+L) Secondary Antibody, Biotin XX, Invitrogen – ThermoFisher, B2770)
• Pierce™ Streptavidin Poly-HRP (ThermoFisher, 21140)
• Plastic sheet protector
• Chemiluminescent substrate (Cell Signaling Technology, SignalFire™ ECL Reagent #6883 or SignalFire™ Elite ECL Reagent #12757)

Method

Day 1

1. Prepare protein samples (10-40µg) in LDS sample buffer with reducing agent in a volume of 30µL or less.
2. Set up gel will 1X running buffer with antioxidant.
3. Load samples and run gel at 200V for 20-60min.
   1. During gel run, preheat incubator to 56°C.
4. Transfer proteins using the standard Trans-Blot Turbo protocol
   1. 25V, 1A, 30 min
   1. During transfer prepare cold acetone in glass trays on ice
   1. During transfer prepare 5% milk and 5% BSA solutions in 1X TBS-T
      1. Each membrane requires 10mL milk and 50mL BSA
5. No-Stain labeling and imaging of membrane
   1. Prepare staining solution
      1. 9.5mL deionized water
      1. 0.5mL 20X No-Staining Labeling Buffer
      1. 20µL No-Stain Activator
      1. 20µL No-Stain Derivatizer
   1. Place 10mL staining solution into a glass tray and add membrane with transferred proteins
   1. Rock slowly (~60rpm) for 10 minutes at room temperature.
   1. Discard labeling solution
   1. Wash membrane with deionized water 3×2 minutes with slow rocking.
   1. Discard final wash solution and replace with deionized water
   1. Place membrane in imager and illuminate with blue light; image using the orange DNA filter
6. Place membrane in cold acetone and incubate for 30 minutes with gentle rocking
7. Pour off acetone in beaker in fume hood
8. Place trays in preheated incubator and incubate for 15-30 minutes until membranes are completely dry
   1. After first 1-2 minutes, open the door to vent acetone vapor
9. Rinse membranes quickly with methanol and place in small plastic lidded boxes with 10mL 5% milk
10. Incubate membrane at room temperature for 60 minutes with gentle rocking.
    1. During incubation prepare 10mL 0.1 mg/mL streptavidin and 10mL 0.5mg/mL biotin blocking solutions in 5% BSA.
11. Wash membrane 3 x 5 minutes with vigorous rocking
12. Incubate membrane in 10mL streptavidin blocking solution at room temperature for 15 minutes with gentle rocking
13. Wash membrane 3 x 5 minutes with vigorous rocking
14. Incubate membrane in 10mL biotin blocking solution at room temperature for 60 minutes with gentle rocking
    1. During incubation, prepare primary antibody dilution (1:1000 – 1:5000) in 10mL 5% BSA
15. Wash membrane 3 x 5 minutes with vigorous rocking
16. Incubate membrane in primary antibody solution at 4°C overnight with gentle oscillation.

Day 2

1. Wash membrane 3 x 5 minutes with vigorous rocking
   1. During washes, prepare secondary antibody dilution (1:5000 – 1:10000) in 10mL 5% BSA
2. Incubate membrane in secondary antibody solution at room temperature for 60 minutes with gentle rocking
   1. During incubation prepare streptavidin-PolyHRP dilution (50 – 100ng/mL) in 10mL 5% BSA
3. Wash membrane 3 x 5 minutes with vigorous rocking
4. Incubate membrane in streptavidin-PolyHRP solution at room temperature for 60 minutes with gentle rocking
5. Wash membrane 3 x 5 minutes with vigorous rocking
6. Transfer membrane to small plastic box
7. Prepare chemiluminescent substrate (0.5mL per membrane) and apply to membrane.
8. Manually rock membrane for 30 seconds to coat with substrate
9. Transfer membrane to plastic sheet protector and roll off bubbles and excess.
10. Take a 25-image stack using the imager with no filter and 2X to 4X camera binning
    1. Start with a 5-second exposure time and adjust up or down as necessary
11. Before removing the membrane capture a white light illuminated image of the membrane (you’ll need to a low exposure and low brightness setting to avoid washout) for later overlay of the ladder onto the blot image.
    1. Optional – to have a color image of the ladder for future reference, take 3 images with white illumination through red, green, and blue filters, false color them to their associated filter, and digitally merge them.