Western blot (SDS–PAGE → semi-dry transfer → immunodetection) — step-by-step protocol

Overview: 

Western blotting (Immuno-Blotting) is a technique which allows to detect specific proteins (antigens) via the interaction with a corresponding antibody. Therefore, proteins which were first separated by their molar mass on a polyacrylamide gel are transferred to a nitrocellulose or polyvinylidenfluorid  (PVDF) membrane by applying an electric field. The membrane is then exposed to a primary antibody solution where the antibody binds solely to the desired protein. Next, the membrane is incubated with a secondary antibody which subsequently binds to the first antibody. The secondary antibody carries an enzyme which catalysis a chemical reaction when exposed to a detection solution resulting in a fluorescent signal that can be measured. Western blotting allows to detect protein amounts below the nanogram level

Material and Chemicals you will need: 

Rotiblock	A151.3	Carl Roth
Amersham ECL Prime Western Blotting Detection Reagent	RPN2236	Sigma-Aldrich
Roti®-PVDF, Porengröße 0,45 μm Bind.hydrophob, 375x26,5cm	T830.1	Carl Roth
Nitrocellulose Porengröße 0,45 µ - 30x400 cm	9201.1	Carl Roth
Whatman Paper	FT2520580600K	Labshop online

10x Semidry-Transfer buffer 

	Company	concentration	1l	2l
Tris	Roth, AE15.3	0,25M	30,3g	60,6g
Glycine	Roth, 3908.3	1,92M	144g	288g

1x Semidry-Transfer buffer

	1l
10x Semidry-Transfer buffer	100ml
Methanol	200ml
ddH2O	700ml

TBS-Puffer 10x, pH 7,4 + 1% Tween 

		c	1l	2l	5l
Tris	Roth, AE 15.3	50mM	60,55 g	121,1 g	302,75g
NaCl	Sigma, S7653	146mM	85,5 g	171 g	427,5g
Tween 20 (20%)		0,1%	50 ml	100 ml	250ml

 

Protocol: 

1) Sample preparation

1. Calculate volume of 4× Laemmli buffer required to dilute each lysate to 1× final.

   • Example: to 3 parts lysate add 1 part 4× Laemmli (total 4 parts) → final = 1×.

2. Add reducing agent if not already present (your 4× has β-ME). Mix gently.

3. Vortex samples 5–10 seconds.

4. Incubate samples at 95°C for 7 minutes (heating block).

5. Remove samples from heat and keep on ice (or at room temp) until loading.

6. Thaw marker on ice.

Notes

• Typical protein load per lane: 10–30 µg total protein (adjust by antibody sensitivity). Use fixed volume if your assay is volume-based.

• Always include a lane map and label tubes clearly.

  
  

  
  

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2) Gel setup & electrophoresis

1. Assemble gels in gel chamber. Make sure gels sit tight at the upper edge of the rubber seal — if not, push them up.

2. Fill inner chamber with 1× SDS running buffer (it can overflow a bit). Wait ~5 minutes and verify no leaks.

3. Fill outer chamber up to the level of the clamps.

4. Load samples and marker:

   • Marker (3.5 µL) always in the leftmost lane.

   • Load your samples into the appropriate wells (record volumes).

5. Close lid and connect electrodes properly.

6. Run gel:

   • 15 min at 90 V until a dye front/band migrates into the stacking gel.

   • Then 130 V for 45–70 min, or until the dye front/blue front approaches the bottom (monitor progress; “until blue clouds emerge from the gel” per your note).

Tip: If you need high resolution for low-MW bands, run longer at lower voltage.

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3) Prepare transfer materials while gel runs

• Cut Whatman paper to ~8.5 × 7.5 cm (4 sheets per blot) and place in bowl with semidry transfer buffer.

• Prepare 3 bowls:

  1. Whatman papers in semidry buffer.

  2. Semidry buffer for gels.

  3. Semidry buffer for membranes.

• Prepare membranes:

  • PVDF: wet in methanol 2 min (can be longer), then rinse in ddH₂O, then equilibrate in transfer buffer.

  • Nitrocellulose: soak directly in ddH₂O briefly (2 min) then transfer buffer.

• Label membranes (do not touch membrane surface; wear gloves).

  
  

  
  

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4) Gel removal and equilibration

1. Turn off power, unplug, open chamber, and pour SDS buffer out of the large chamber.

2. Use the green spatula to separate glass plates carefully.

3. Remove and discard the stacking gel (cut off) — keep resolving gel.

4. Place gel in bowl with semidry transfer buffer in the correct orientation (this is the orientation the membrane will lie on the gel).

5. Move membranes from methanol → ddH₂O → transfer buffer bowl (labeled side up). Ensure membranes do not touch when storing >1 membrane.

6. Gently agitate bowls to soak components; incubate 5 minutes.

7. Clean the SDS chamber with soap and let dry (if applicable) while you finish equilibration.

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5) Assemble semidry transfer “sandwich”

Orientation is critical (from bottom/up relative to anode/cathode and your unit — follow instrument manual). The membrane should sit directly on the gel on the side facing the cathode → anode transfer direction.

1. Place two pre-soaked Whatman papers (stacked) on the semidry unit plate.

2. Remove trapped air by rolling the plastic tube along the papers lengthwise and widthwise.

3. Place the membrane on top of the papers (labeling must be legible; place the membrane face up so the protein side faces the gel). Avoid air bubbles — if bubbles occur, lift and re-seat until gone.

4. Place the gel on top of the membrane in the exact orientation it was in the gel cassette.

5. Gently remove any remaining air bubbles with the wet spatula (do not scratch the gel).

6. Place two more Whatman papers on top of the gel.

7. Roll the tube again to expel air.

8. Close the lid, place two water bottles (or weights) on top to ensure good contact.

9. Connect and set transfer parameters:

   • Constant current mode: 0.07 A (70 mA) per blot.

   • Time: 90 minutes.

(These are the parameters you provided — if you usually use different amperage/time for PVDF vs nitrocellulose, follow your lab standard.)

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6) Post-transfer processing

1. Disassemble chamber, remove membrane carefully.

2. Mark molecular weight marker on the membrane with a pen on the membrane border (not where antibodies will bind).

3. Optional: stain membrane briefly with Ponceau S to confirm transfer and orientation; then wash with TBST or ddH₂O to remove stain.

   
   

   
   

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7) Blocking & primary antibody incubation

1. Block membrane in blocking solution (e.g., 5% milk in TBST or 3% BSA in TBST) 45 minutes at room temperature with gentle agitation.

2. Incubate membrane in primary antibody diluted in blocking solution (use your antibody’s recommended dilution).

   • Incubate overnight at 4°C with gentle shaking.

Notes

• Record the exact dilution and volume (e.g., 1:1000 in 10 mL blocking solution).

• Use an appropriate container to fully cover membrane.

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8) Washing and secondary antibody

1. Next day: wash membrane 3 × 5–10 minutes in TBST with agitation (room temp).

2. Incubate membrane in secondary antibody diluted in blocking solution (use recommended dilution) for 1 hour 45 minutes at room temperature with gentle agitation.

3. Wash membrane 3 × 5–10 minutes in TBST after secondary incubation.

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9) Detection

1. Mix ECL detection solutions 1:1 immediately before use (as per your reagent instructions).

2. Apply ECL solution to the membrane, ensuring full coverage.

3. Place membrane in autoradiography cassette or imaging system and image using appropriate exposure(s) on your chemiluminescence camera.

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10) Imaging & data handling

• Acquire multiple exposure times to avoid saturation.

• Save raw image files and processed images separately. Record antibody lot numbers, dilutions, exposure times and any image processing steps.

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Troubleshooting tips

• No bands / weak signal: check transfer efficiency (Ponceau stain), antibody activity/dilution, sufficient protein load, ECL working.

• High background: increase blocking time or change blocker (milk → BSA), reduce primary/secondary concentration, increase wash stringency (more washes or longer).

• Smearing or fuzzy bands: overloaded lane, degraded sample, incomplete reduction/boiling, or salt in samples — clean up lysates.

• Air bubbles during transfer: will cause white spots (no signal) — always roll/tube to remove bubbles.

• Uneven transfer: ensure even contact, clean and flat papers, correct orientation, and proper current setting.

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Record-keeping checklist (log for each blot)

• Date, operator.

• Gel % and manufacturer.

• Sample IDs and amounts loaded per lane.

• Marker used and volume (you stated 3.5 µL).

• Transfer membrane type and lot, methanol step (if PVDF).

• Transfer settings: current (0.07 A/blot), time (90 min).

• Blocking reagent, primary and secondary antibodies (vendor, catalog, lot), dilutions, incubation times/temps.

• ECL reagent and exposure times.

• Imaging device and settings.