- Polyacrylamide gels (% depends upon target protein molecular weight)
- Molecular Weight markers for visualization on gel: Precision Plus Protein Dual Color Standard – Biorad 161-0374
- Molecular weight markers for visualization on membrane: MagicMark XP Western Standard – Invitrogen LC5602
- Sample Buffer (Loading Dye): NuPAGE LDS Sample Buffer (4X) – Invitrogen NP0007
- NuPAGE Sample Reducing Agent (10x) – Invitrogen NP0004
- NuPAGE Antioxidant – Invitrogen NP0005
- SDS-Page Running Buffer: NuPage MES SDS Running Buffer (20X) - Invitrogen NP0002
- 1x Transfer Buffer: NuPage Transfer Buffer (20X) - Invitrogen NP0006-1
- PVDF Membrane Filter Paper 0.2 μm pore - Invitrogen LC2002
- Filter Paper
- Foam sponges
Western Blot and Visualization
- Primary antibody: can be raised in rabbit, mouse, chicken, goat, sheep, etc.
- HRP conjugated secondary antibody raised against the primary
- 1 X TBS-Tween Buffer
- Tween-20 - Biorad # 170-6531
- Blocking solution: TBS + Tween-20 + BSA Fraction V (Sigma Chemical Co. # A-2153) + dH2O
- Detection Reagent: Western ECL detection: Pierce (Dura West)
Protein Separation by SDS-PAGE
- Running Buffer: Add 50 ml 20X concentrate to 850ml dH2O
- Loading Samples: Add 8μl of sample (at concentration of 1mg/ml), 1μl Reducing Agent (10X), and 2.5μl LDS Sample Buffer. MAINTAIN THIS RATIO if loading a different amount
- Place gel into the core and put into tank. Wells should face the inside for loading.
- Close apparatus and use running buffer to make sure that there are no leaks.
- Add Buffer to the tank and inside the core
- Add 500μl Antioxidant to the middle of the tank
- Fill wells with loading samples. Leave space for the molecular weight markers. If you are running a western on the SDS-PAGE, leave a space in between sample groups
- Run @ 100 volts (30 mA) until dye line runs off the gel.
Protein Transfer to PVDF
- Dilute transfer buffer to 1x and add 100ml MeOH - store @ 4°C. For NuPAGE Novex Bis-Tris or Tris-Acetate Gels: 50ml NuPage Transfer Buffer (20x), 100ml methanol, 850ml dH2O
- Prepare a dish of MeOH and of transfer buffer for PVDF
- Soak pads in cold transfer buffer
- Dip both sides of the membrane in MeOH, water, and soak in cold transfer buffer
- Load transfer apparatus
- Dip filter paper in TB before putting on apparatus
- Put 2 pads/side.
- Remove gel from its case. Add filter paper and gel to top of pads
- Add PVDF making sure there are no air bubbles and another piece of filter paper. Use a small glass test tube to gently roll out air bubbles. Complete the set up.
- Place inside transfer apparatus and fill with cold transfer buffer - make sure it does not leak and do not fill all the way to the top. Add COLD H2O or TB to outside
- Set up transfer apparatus on ice (or in cold room). For Bis-Tris gels, run @ 30 V volts for 1 hr. Expected current starts at 170mA and ends at 110mA.
- Remove membrane paper from transfer buffer apparatus. Wet in MeOH and dry on filter paper overnight
- Wash Buffer: 1x TBS-T - Add 5ml of 10% Tween-20 per liter of TBS (final concentration ~0.05%)
- Blocking buffer: Combine 100 ml TBS, 10 ml Tween-20, 10 g BSA fraction V, and 890 ml dH2O. AVOID using milk when using Avidin/biotin systems.
- Primary Antibody: For SuperSignal West Dura Extended Duration Substrate, the concentration of primary antibody needs to be between 20ng/ml and 1μg/ml. Dilute in blocking buffer (1:1000 = 10 μl per 10 ml) - for a 1mg/ml antibody solution, dilutions should be in the range of 1:1,000 to 1:50,000.
- Secondary antibody: For SuperSignal West Dura Extended Duration Substrate, the concentration of secondary antibody needs to be between 4-20ng/ml. Dilute in blocking buffer (1:50,000 = 1 μl per 50ml) - for a 1mg/ml antibody solution, dilutions should be in the range of 1:50,000 to 1:250,000.
- Detection regent: 10 ml enhancer solution + 10 ml peroxide (DO NOT MIX UNTIL READY; 1:1 ratio)
- Reactivate in MeOH
- Block: place membrane in blocking roughly 50 ml buffer for 1.5 hours at room temperature or overnight @ 4°C. Incubate on a rocker platform.
- Dilute primary antibody in blocking buffer and incubate according to manufacturer's instructions. Incubate on a rocker platform. Incubate 1st @ 4°C overnight
- Wash blot 4-6 X 5 minutes in Wash Buffer on a rocker platform
- Prepare secondary antibody in blocking buffer (12ml for one blot). Incubate on a rocker platform.
- Place membrane in secondary antibody solution @ RT for 1 hr.
- Additional blocking agents may be added if background staining is an issue.
- Wash 4-6 X 5 min in Wash Buffer on a rocker platform
- Mix together detection reagent and soak membrane for 5 minutes
- Rock manually or on rocking platform and observe membrane while color development takes place.
- Optional: Stop color development when bands are easily visualized or when background color development begins to be excessive, by decanting the development solution into a waste container, and adding approximately 50 ml of deionized water to the incubation tray.
- Optional: Return the tray to the rocking platform and check the color of the water after 5-10 minutes. Decant and replace with fresh water if there is a significant purple tinge to the water, or if band intensity has continued to increase.
- Place blot on sheet protector and make sure the outside is clean and the inside is bubble free. Remove excess liquid/Keep in dark
- Visualize blot using x-ray film or chemiluminescent gel imager