Western Blot (SDS-PAGE and Immunoblotting)

Western Blot (SDS-PAGE and Immunoblotting)

biochemistry · Intermediate · 2–3 days (8–10 hr active; overnight primary antibody incubation required)

https://labgrimoire.com/protocols/western-blot-sds-page/

Materials

• ☐ Acrylamide/Bis solution (30%, 29:1 crosslinker ratio): Variable (⚠ Neurotoxic in unpolymerized form; handle with nitrile gloves in a fume hood. Store at 4°C, protect from light.)
• ☐ 1.5 M Tris-HCl, pH 8.8: Variable (Resolving gel buffer. Prepare in ddH₂O, adjust pH with HCl, autoclave or filter-sterilize, store at 4°C.)
• ☐ 1.0 M Tris-HCl, pH 6.8: Variable (Stacking gel buffer. Prepare and store as above.)
• ☐ SDS, 10% (w/v) solution: Variable (Sodium dodecyl sulfate in ddH₂O. Store at room temperature; may precipitate at < 15°C — warm and dissolve before use.)
• ☐ APS, 10% (w/v) ammonium persulfate: Variable (Prepare fresh in ddH₂O or aliquot and store at −20°C for up to 2 months. Activity degrades significantly after 2–4 weeks at 4°C.)
• ☐ TEMED (N,N,N′,N′-tetramethylethylenediamine): Variable (Polymerization catalyst. Store at 4°C. Discard if color darkens or odor changes.)
• ☐ 2× or 4× Laemmli SDS sample buffer: Variable (Contains SDS, glycerol, Tris-HCl pH 6.8, bromophenol blue. Add β-mercaptoethanol fresh before each use.)
• ☐ β-Mercaptoethanol (β-ME): 50–100 μL per experiment (Reducing agent for disulfide bond cleavage. Volatile, malodorous — add only in a chemical fume hood.)
• ☐ Pre-stained protein molecular weight ladder: 3–5 μL per lane (Choose a ladder whose MW range brackets the expected size of your target protein.)
• ☐ Isopropanol (100%): ~5 mL (Overlay resolving gel during polymerization to exclude oxygen.)
• ☐ Methanol (100%): 100 mL (PVDF membrane activation and transfer buffer preparation.)
• ☐ PVDF membrane (0.45 μm pore size): As needed (Standard choice for most targets. Use 0.22 μm pore size for proteins < 20 kDa.)
• ☐ Nitrocellulose membrane: As needed (Alternative to PVDF. Does not require methanol activation.)
• ☐ Transfer buffer (1×): 1 L (25 mM Tris, 192 mM Glycine, 20% (v/v) methanol in ddH₂O. Do not add SDS unless transferring high-MW proteins. Pre-chill to 4°C.)
• ☐ 10× TBS (Tris-buffered saline): Variable (200 mM Tris-HCl pH 7.6, 1.5 M NaCl in ddH₂O. Dilute to 1× and add Tween-20 to 0.1% (v/v) to make TBS-T.)
• ☐ Blocking buffer — milk: 10–20 mL (5% (w/v) non-fat dry milk dissolved in TBS-T. Prepare fresh. Use for standard (non-phosphorylated) protein targets.)
• ☐ Blocking buffer — BSA: 10–20 mL (5% (w/v) bovine serum albumin dissolved in TBS-T. Prepare fresh. Use for phospho-specific antibody targets.)
• ☐ Primary antibody (target-specific): Variable (Dilute in appropriate blocking buffer per manufacturer's datasheet.)
• ☐ HRP-conjugated secondary antibody: Variable (Species-matched to primary antibody (anti-rabbit, anti-mouse, etc.).)
• ☐ ECL chemiluminescence substrate: Variable (Two-reagent system (Reagent A + Reagent B). Mix 1:1 immediately before use.)
• ☐ Ponceau S staining solution: 5 mL (0.1% (w/v) Ponceau S in 5% (v/v) acetic acid. Reusable; store at room temperature.)
• ☐ 1× Running buffer (Tris-Glycine-SDS): 1 L (25 mM Tris, 192 mM Glycine, 0.1% SDS in ddH₂O.)
• ☐ Stripping buffer (optional): 10–20 mL (62.5 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 100 mM β-ME. Prepare in a fume hood immediately before use.)

Equipment

• ☐ Mini SDS-PAGE apparatus (glass plates, spacers 1.0 mm, comb) (Standard mini-gel format (~8 × 7 cm). Ensure plates are clean and grease-free.)
• ☐ Electrophoresis power supply (Constant-voltage mode; capable of 80–120 V.)
• ☐ Wet transfer apparatus (transfer tank, cassette, fiber pads) (Fill with pre-chilled transfer buffer; include a magnetic stir bar for buffer circulation.)
• ☐ Cooling device for transfer (ice pack or recirculating cooler) (Maintains buffer at 4°C during high-current transfer to prevent gel swelling and heat artifacts.)
• ☐ Chemiluminescence CCD imaging system (Capable of multiple timed exposures and 16-bit image capture for densitometry. Film-based detection is acceptable but is less quantitative.)
• ☐ Rocking or oscillating platform (rocker) (For all antibody incubations and membrane washes.)
• ☐ Heating block or water bath (set to 95–100°C or 37°C) (For protein sample denaturation.)
• ☐ Microcentrifuge (For brief centrifugation of denatured samples (10,000 × g, 30 sec).)
• ☐ Spectrophotometer or plate reader (595 nm, for Bradford assay) (For protein quantification; alternatively, BCA microplate format.)
• ☐ Flat-tip forceps (For membrane handling. Never touch the membrane with bare hands.)
• ☐ Glass rod or 15 mL conical tube (For rolling out air bubbles when assembling the transfer sandwich.)
• ☐ Filter paper (cut to gel size) (To pad the transfer sandwich; soak in transfer buffer before assembly.)

Procedure

1. ☐ Select acrylamide concentration and prepare resolving gel solution (10 min)

   — Part A: Gel Preparation —

   Determine the appropriate resolving gel acrylamide concentration based on the expected molecular weight of the target protein:

   Target MW	Recommended acrylamide %
   > 100 kDa	8%
   40–100 kDa	10%
   25–60 kDa	12%
   10–30 kDa	15%

   Prepare 10 mL of resolving gel solution by combining the following reagents in order in a 50 mL conical tube (example for 10% gel):

   Component	8%	10%	12%	15%
   ddH₂O	4.6 mL	4.0 mL	3.3 mL	2.3 mL
   30% Acrylamide/Bis (29:1)	2.7 mL	3.3 mL	4.0 mL	5.0 mL
   1.5 M Tris-HCl pH 8.8	2.5 mL	2.5 mL	2.5 mL	2.5 mL
   10% SDS	100 μL	100 μL	100 μL	100 μL

   Mix gently by swirling. Do not add APS or TEMED at this stage.

2. ☐ Cast resolving gel and allow to polymerize (30–45 min)

   Assemble the gel casting apparatus according to manufacturer instructions. Verify no leaks by filling with water before casting. Add to the resolving gel solution, in order:

   • 10% APS: 100 μL
   • TEMED: 4 μL (for 10%; use 6 μL for 8%, 4 μL for 12% and 15%) Mix gently by swirling (do not vortex — introduces air bubbles). Immediately pour the gel mixture between the glass plates to approximately 1.5 cm below the top edge of the short plate. Gently overlay with ~500 μL of 100% isopropanol. Allow to polymerize at room temperature for 30–45 minutes. A sharp, refractive phase boundary visible just below the isopropanol layer confirms complete polymerization.
3. ☐ Prepare stacking gel and cast onto resolving gel (15–20 min)

   Discard the isopropanol overlay completely; blot residual liquid at the gel surface with a strip of filter paper. Prepare 5 mL of 5% stacking gel solution:

   Component	Amount
   ddH₂O	3.4 mL
   30% Acrylamide/Bis (29:1)	830 μL
   1.0 M Tris-HCl pH 6.8	630 μL
   10% SDS	50 μL

   Add 50 μL 10% APS and 5 μL TEMED; mix gently by swirling. Immediately pour stacking gel solution onto the resolving gel surface, filling to the top of the glass plates. Insert the well comb smoothly, avoiding air bubble trapping under comb teeth. Allow to polymerize at room temperature for 15–20 minutes.

4. ☐ Quantify protein and prepare equalized samples (30 min)

   Determine protein concentration of each sample using a Bradford or BCA assay. Calculate the volume of each sample required to deliver an equal protein mass per lane. A typical loading amount is 20–40 μg total protein per lane; adjust based on target protein abundance. Combine each sample volume with 2× Laemmli sample buffer (add equal volume of 2× buffer) or 4× buffer (add one-third volume of 4× buffer). Add β-mercaptoethanol to the sample buffer at 5% (v/v) final concentration; perform this step in a chemical fume hood. Verify that all lanes receive the same final volume after normalization.

5. ☐ Denature protein samples (5 min)

   Standard soluble proteins: Heat samples in a heating block at 95–100°C for 5 minutes. Integral membrane proteins (multi-pass transmembrane proteins): Heat at 37°C for 30 minutes to prevent heat-induced irreversible aggregation and high-MW smearing. Centrifuge samples at 10,000 × g for 30 seconds to pellet any insoluble debris and collect condensate. Keep samples on ice until loaded onto the gel.

6. ☐ Load gel and run SDS-PAGE electrophoresis (80–120 min)

   Mount the polymerized gel cassette in the electrophoresis tank; fill inner and outer chambers with 1× running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS). Carefully remove the well comb; flush wells with running buffer using a gel-loading tip to remove any unpolymerized acrylamide or debris. Load 3–5 μL of pre-stained molecular weight ladder into the first lane. Load protein samples into subsequent wells using a gel-loading tip, inserting to just above the well bottom to avoid bubble formation. Run at 80 V (constant voltage) until the bromophenol blue dye front migrates through the stacking gel and enters the resolving gel (approximately 20–30 min). Increase voltage to 120 V and continue until the bromophenol blue front reaches the bottom of the resolving gel (approximately 60–90 min). Stop electrophoresis when the bromophenol blue front is 0.5 cm from the bottom edge of the gel.

7. ☐ Activate PVDF membrane and assemble transfer sandwich (10 min)

   Cut PVDF membrane and filter paper sheets to the dimensions of the gel. PVDF membrane: Immerse in 100% methanol for exactly 15 seconds (until it changes from white to semi-transparent), then transfer immediately to transfer buffer for ≥5 minutes equilibration. Nitrocellulose membrane (alternative): Skip methanol step. Immerse directly in transfer buffer for 5 minutes. Pre-chill 1 L of transfer buffer (25 mM Tris, 192 mM Glycine, 20% v/v methanol in ddH₂O) to 4°C.

   • For proteins > 100 kDa: use transfer buffer with 10% methanol + 0.05% SDS to improve transfer efficiency for large proteins. Assemble the transfer sandwich in a tray of transfer buffer (from cathode [black face] to anode [red face]):

   [Cathode / Black] → Fiber pad → Filter paper → Gel → Membrane → Filter paper → Fiber pad → [Anode / Red]
   

   Roll over the assembled sandwich with a glass rod to eliminate all air bubbles between gel and membrane.

8. ☐ Run wet electroblotting transfer (60–90 min)

   Insert the assembled sandwich cassette into the transfer tank, ensuring gel faces the cathode (black connector) and membrane faces the anode (red connector). Fill the transfer tank completely with pre-chilled transfer buffer; add a magnetic stir bar for buffer circulation. Surround the transfer tank with ice or place in a cold room at 4°C. Run transfer at 100 V (constant voltage) for 60–90 minutes.

   • For proteins > 150 kDa: extend to 120 minutes, or transfer overnight at 30 V, 4°C.
9. ☐ Verify transfer efficiency with Ponceau S staining (10 min)

   Disassemble the transfer cassette; remove the membrane with forceps. Immerse membrane in Ponceau S staining solution (0.1% w/v in 5% acetic acid) for 5 minutes with gentle agitation. Rinse with ddH₂O until protein bands appear as pink/red bands against a white background. Verify: bands are present across all sample lanes; ladder is visible; no white circular voids (bubble artifacts); distribution looks uniform. Photograph the Ponceau-stained membrane for records. Fully destain the membrane by washing with TBS-T (3 × 5 min) before proceeding to blocking.

10. ☐ Block membrane to reduce non-specific antibody binding (1 hr)

    Select blocking buffer based on antibody target:

    • 5% non-fat dry milk in TBS-T → for standard protein targets (e.g., total protein levels, structural proteins).
    • 5% BSA in TBS-T → for phospho-specific antibodies (phosphorylated protein targets) or any antibody that the datasheet specifies should not use milk. Prepare blocking buffer fresh by dissolving the blocking agent in 1× TBS-T at room temperature. Immerse membrane in blocking buffer; incubate at room temperature for 1 hour with gentle rocking.
11. ☐ Incubate with primary antibody (overnight, 4°C)

    Dilute primary antibody in the appropriate blocking buffer at the concentration recommended in the manufacturer's datasheet. A typical starting dilution for HRP/ECL detection is 1:500 to 1:2,000 (optimize empirically for each antibody). Transfer membrane to a sealed container (hybridization bag, small box, or resealable bag) with the primary antibody solution, ensuring complete membrane coverage with minimal antibody volume. Incubate at 4°C overnight (12–16 hours) with gentle continuous rocking.

12. ☐ Wash membrane to remove unbound primary antibody (30 min)

    Pour off primary antibody solution. If desired for reuse, recover it into a clean tube and store at 4°C with 0.02% sodium azide (typically stable for 2–3 reuse cycles depending on antibody). Wash membrane three times with TBS-T: 10 minutes per wash, room temperature, with vigorous rocking. Discard wash solution after each wash.

13. ☐ Incubate with HRP-conjugated secondary antibody (1 hr)

    Dilute HRP-conjugated secondary antibody in 5% non-fat dry milk / TBS-T at 1:5,000 to 1:10,000 (optimize for each lot/antibody).

    • Use milk blocking buffer for the secondary antibody even when the primary antibody required BSA blocking. Incubate membrane with secondary antibody solution at room temperature for 1 hour with gentle rocking.
14. ☐ Wash membrane to remove unbound secondary antibody (30 min)

    Wash membrane three times with TBS-T: 10 minutes per wash, room temperature, with vigorous rocking. After the final wash, blot the membrane edge lightly against a paper towel to remove excess buffer, but do not allow the membrane to dry.

15. ☐ Apply ECL substrate and acquire chemiluminescence images (15 min)

    Prepare ECL substrate immediately before use: mix Reagent A and Reagent B in equal volumes per manufacturer's instructions. Use only the minimum volume needed to cover the membrane surface. Transfer membrane to a clean, flat surface (protein-side up); pipette ECL substrate evenly across the entire membrane surface. Incubate for the time specified by the manufacturer (typically 1–5 minutes). Drain excess substrate by tilting the membrane; do not rinse. Transfer membrane to the imaging system and acquire images at multiple exposure times (e.g., 5 sec, 15 sec, 30 sec, 1 min, 5 min). Select the shortest exposure time that yields the maximum unsaturated signal for densitometric analysis.

16. ☐ Perform densitometric quantification and statistical analysis (30 min)

    Open the selected unsaturated image in ImageJ (FIJI), Image Lab, or equivalent densitometry software. Define a consistent region of interest (ROI) around each band in each lane; apply background subtraction using a consistent method (rolling ball algorithm or adjacent lane background). Measure the integrated band intensity (optical density × area, in arbitrary units) for each target protein band. Measure the integrated band intensity for the loading control protein in the same lane (e.g., β-actin at 42 kDa, GAPDH at 37 kDa, or α-tubulin at 50 kDa).

    • Select a loading control that does not co-migrate with the target protein band (avoid same-MW overlap). Calculate normalized protein expression per lane using:

    Normalized intensity = (Target protein band intensity) ÷ (Loading control band intensity) Express results as fold change relative to the control group (set control group mean to 1.0). Collect data from ≥3 independent biological replicates (each replicate is a separately prepared and independently run experiment; technical replicates within a single experiment do not substitute for biological replication). Perform statistical analysis: Student's t-test (two-group comparison) or one-way ANOVA with post-hoc test (three or more groups). Report mean ± SEM or SD with sample size (n).

17. ☐ Strip membrane and reprobe with a different antibody (50 min + 10 min wash)

    Prepare stripping buffer in a chemical fume hood: 62.5 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 100 mM β-mercaptoethanol in ddH₂O. Transfer membrane to stripping buffer in a container that can be sealed; incubate at 50°C for 30 minutes with gentle agitation inside the fume hood. Transfer membrane to a tray of TBS-T; wash 3 × 10 minutes at room temperature to remove stripping buffer. Perform a brief ECL exposure to confirm complete removal of the previous antibody signal before reprobing. Proceed to blocking (Step 10) and repeat immunodetection (Steps 11–15) with the new antibody.