Oct 06, 2025
- igemnthuth 1
- 1iGEM_NTHU_Taiwan
- iGEM NTHU
Protocol Citation: igemnthuth 2025. WB. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4mndlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 06, 2025
Last Modified: October 06, 2025
Protocol Integer ID: 229086
Keywords: western blot, wb, protein, protein expression
Abstract
We use Western blot to quantify and verify the protein’s expression level.
Materials
- Vortex mixer, microcentrifuge, rocking platform/shaker
- Dry heating block at 95 °C (metal dry bath)
- Deionized water (MQ)
- Forceps, roller/glass rod, scissors/utility knife, straightedge/plate clip
- Shallow trays/dishes (for membrane incubation and washes)
- Insulated box/cooler (polystyrene), ice and ice packs
- PPE: lab coat, gloves, safety goggles (use fume hood for toxic/volatile reagents)
- Glass plates (large/small), spacers, clamps, comb(s), casting cassette
- Acrylamide solution
- Tris buffers (separate pH for resolving and stacking gels)
- SDS (sodium dodecyl sulfate)
- TEMED
- APS (ammonium persulfate)
- Gel tank and power supply
- Running buffer (e.g., Tris–Glycine–SDS)
- Protein loading dye (e.g., Laemmli sample buffer; final 1× in samples)
- Protein samples
- Protein molecular-weight marker/ladder
- Transfer tank and cassette (cathode/anode sides)
- Transfer buffer
- Sponges ×2
- Filter papers ×6
- Membrane ×1 (PVDF or nitrocellulose)
- Methanol (for PVDF activation)
- TBST (TBS with Tween-20)
- Nonfat dry milk (to prepare 5% w/v blocking buffer)
- Primary antibody (diluted per datasheet in blocking/dilution buffer)
- Secondary antibody (typically HRP-conjugated)
- ECL chemiluminescent substrate
- Chemiluminescence imager/CCD system
- 96-well clear flat-bottom microplate
- Protein assay dye (e.g., Bradford-type or compatible)
- Protein standard (≥5 concentration points)
- Microplate reader (commonly 595 nm)
- (Optional) Multichannel pipette
Troubleshooting
SDS-PAGE Gel Casting
Place the cassette upside down; insert spacers and mount the small front and large back glass plates. Fix tightly with clamps.
Add a little MQ water to check for leaks. If leak-free, discard the water and blot completely dry with filter paper.
In a conical tube, add in order: acrylamide → Tris (resolving pH) → SDS. Vortex to mix. Do not add TEMED/APS yet.
Overlay ~800 µL MQ water on the gel surface to exclude air.
Polymerize ≥30 min (recommended 60 min) until a clear interface line appears. Decant the overlay and blot the surface dry.
In a Just before casting, add TEMED and APS, mix briefly, and pour on top of the resolving gel.second tube, add in order: acrylamide → Tris (stacking pH) → SDS; vortex.
Just before casting, add TEMED and APS, mix briefly, and pour on top of the resolving gel.
Immediately insert the comb, aligning the teeth with the top edge of the small plate; remove any trapped bubbles.
Allow to polymerize 20–30 min. Remove the comb; the gel is ready for loading.
96-Well Plate Protein Quantification
Prepare a 5-point standard curve from high to low (include 0 point), within the kit’s linear range. If sample concentrations are unknown, pre-dilute to ensure readings fall within the linear range.
Dispense the designated volume of standard/sample into assigned wells.（Keep the pipette tip below the liquid surface and dispense along the wall to minimize bubbles and splashing.）
Add Dye to each well.
Read absorbance at the specified wavelength on a plate reader.
Build the standard curve from blank-subtracted absorbance of the standards.（Accept the curve only if R² ≥ 0.95.）
SDS-PAGE Electrophoresis
Add loading dye to the final desired concentration , scaling with the planned load volume). Heat at 95 °C for 7 min, then briefly spin down.
Mount the gel cassette on the apparatus. Level and secure; align the top edge with the reference mark.
Slowly add running buffer to the inner chamber first, ensuring all well bubbles are expelled. Add the remaining buffer to the outer chamber. Keep the buffer level above the top edge of the small plate throughout the run.
Load samples (and marker) with the tip below the liquid surface, dispensing along the well wall to avoid rupturing wells and forming bubbles.
Run at constant current, 35 mA for ~30–40 min (adjust for gel percentage/apparatus). Continuously monitor buffer level; do not let it drop below the small plate edge.
Stop when the tracking dye approaches the gel bottom. Decant buffer, gently pry open the cassette, and remove the gel. Trim off the stacking gel and cut a corner notch near the first well as an orientation mark.
Transfer
Immerse sponges ×2, filter papers ×6, membrane ×1 fully in transfer buffer to pre-soak/equilibrate. If using PVDF, briefly wet in methanol first, then equilibrate in transfer buffer.
Place the transfer unit in an insulated box with ample ice/ice packs to maintain low temperature throughout the run.
Open the cassette and layer in order: Sponge → Filter paper → Gel → Membrane → Filter paper → Sponge. After placing the gel and membrane, gently roll to expel all bubbles and ensure full contact.
Close the cassette firmly (press down and push in to lock). Insert into the tank, confirming black to black (cathode) and red to red (anode) .
Add sufficient transfer buffer to cover the stack and electrodes; add ice packs as needed.
Run at 400 mA for 90 min (adjust for gel thickness/protein size). Do not let the membrane dry at any point.
Disassemble the stack; lift the membrane (keep it wet) with forceps. Trim excess edges using a straightedge, and cut a corner notch near lane 1 for orientation. Proceed immediately to blocking.
Membrane Blocking and TBST Washing
In a 50 mL tube, add 1 g milk powder and bring to 20 mL with TBST; mix until fully dissolved (5% w/v).
Pour blocking buffer into a shallow tray and fully submerge the membrane (mini-gels typically require ~5 mL, adjust to area).
Rock gently at room temperature for 1 h, ensuring the membrane remains fully wet with no dry spots.
Wash 3× with fresh TBST, 1 min each, under gentle rocking. Replace TBST for each wash.
Adding antibodies
Incubate the membrane with the primary antibody (diluted per datasheet in blocking buffer) overnight with gentle rocking at 4 °C (or room temperature as recommended).
The next day, wash with TBST 3×, 10 min each under gentle rocking
Incubate with the appropriate secondary antibody (diluted per datasheet in blocking buffer) for 1 h with gentle rocking at room temperature.
Wash with TBST 3×, 10 min each under gentle rocking.
Add ECL substrate to cover the membrane, incubate briefly per kit instructions, then capture chemiluminescence using an imager. (Avoid drying the membrane; protect from strong light as needed.)