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Western Blot (SDS-PAGE)

Category: Molecular Biology

Materials Required

SDS-Page

  1. Polyacrylamide gels (% depends upon target protein molecular weight)
  2. Molecular Weight markers for visualization on gel: Precision Plus Protein Dual Color Standard – Biorad 161-0374
  3. Molecular weight markers for visualization on membrane: MagicMark XP Western Standard – Invitrogen LC5602
  4. Sample Buffer (Loading Dye): NuPAGE LDS Sample Buffer (4X) – Invitrogen NP0007
  5. NuPAGE Sample Reducing Agent (10x) – Invitrogen NP0004
  6. NuPAGE Antioxidant – Invitrogen NP0005
  7. SDS-Page Running Buffer: NuPage MES SDS Running Buffer (20X) - Invitrogen NP0002

Protein Transfer

  1. 1x Transfer Buffer: NuPage Transfer Buffer (20X) - Invitrogen NP0006-1
  2. PVDF Membrane Filter Paper 0.2 μm pore - Invitrogen LC2002
  3. Filter Paper
  4. Foam sponges

Western Blot and Visualization

  1. Primary antibody: can be raised in rabbit, mouse, chicken, goat, sheep, etc.
  2. HRP conjugated secondary antibody raised against the primary
  3. Methanol
  4. 1 X TBS-Tween Buffer
  5. Tween-20 - Biorad # 170-6531
  6. Blocking solution: TBS + Tween-20 + BSA Fraction V (Sigma Chemical Co. # A-2153) + dH2O
  7. Detection Reagent: Western ECL detection: Pierce (Dura West)
  8. Peroxide

 

Protein Separation by SDS-PAGE

Solutions:

  • 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

Proceedure:

  1. Place gel into the core and put into tank. Wells should face the inside for loading.
  2. Close apparatus and use running buffer to make sure that there are no leaks.
  3. Add Buffer to the tank and inside the core
  4. Add 500μl Antioxidant to the middle of the tank
  5. 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
  6. Run @ 100 volts (30 mA) until dye line runs off the gel.

 

Protein Transfer to PVDF

Solutions:

  • 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

Proceedure:

  1. Prepare a dish of MeOH and of transfer buffer for PVDF
  2. Soak pads in cold transfer buffer
  3. Dip both sides of the membrane in MeOH, water, and soak in cold transfer buffer
  4. Load transfer apparatus
  5. Dip filter paper in TB before putting on apparatus
    1. Put 2 pads/side.
    2. Remove gel from its case. Add filter paper and gel to top of pads
    3. 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.
  6. 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
  7. 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.
  8. Remove membrane paper from transfer buffer apparatus. Wet in MeOH and dry on filter paper overnight

 

Detection

Solutions:

  • 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)

Proceedure:

  1. Reactivate in MeOH
  2. 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.
  3. Dilute primary antibody in blocking buffer and incubate according to manufacturer's instructions. Incubate on a rocker platform. Incubate 1st @ 4°C overnight
  4. Wash blot 4-6 X 5 minutes in Wash Buffer on a rocker platform
  5. Prepare secondary antibody in blocking buffer (12ml for one blot). Incubate on a rocker platform.
    1. Place membrane in secondary antibody solution @ RT for 1 hr.
    2. Additional blocking agents may be added if background staining is an issue.
  6. Wash 4-6 X 5 min in Wash Buffer on a rocker platform
  7. Mix together detection reagent and soak membrane for 5 minutes
    1. Rock manually or on rocking platform and observe membrane while color development takes place.
    2. 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.
    3. 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.
  8. Place blot on sheet protector and make sure the outside is clean and the inside is bubble free. Remove excess liquid/Keep in dark
  9. Visualize blot using x-ray film or chemiluminescent gel imager

Last Edited By: Protocol Monkey on 2009-10-23 [20:41:42]