Protein Tris-Tricine Electrophoresis System Standard Operating Procedure

1. Experimental Equipment

• ertical electrophoresis system: Power supply, electrophoresis tank, and associated accessories.
• Gel casting assembly: Thick plate (spacer plate, 1.0 mm), thin plate (short plate), and plastic frame.
• Pipetting tools: Micropipettes and tips (1 mL, 200 μL, 10 μL).
• Centrifugation and storage consumables: Centrifuge tubes (1.5 mL, 15 mL, 50 mL).
• Glassware: Beakers, volumetric flask (100 mL), graduated cylinders (100 mL, 500 mL, 1000 mL), glass rods.
• Reagent containers: Wide-mouth reagent bottles (500 mL, 1000 mL), amber bottles (250 mL, 500 mL).
• Laboratory consumables: Filter paper, lint-free wipes, plastic wrap/sealing bags.
• Measurement and monitoring: pH meter, etc.

2. Experimental Reagents

1) Ethylene glycol 

2) 40% Acrylamide:N,N′-methylenebisacrylamide (29:1) (40% Acr-Bis) for stacking gel

Component	Amount
Acrylamide	38.67 g
N,N′-methylenebisacrylamide	1.33 g
ddH₂O	Bring to 100 mL

Store at 4°C protected from light after preparation.

3) 40% Acrylamide:N,N′-methylenebisacrylamide (19:1) (40% Acr-Bis) for separation gel

Component	Amount
Acrylamide	38 g
N,N′-methylenebisacrylamide	2 g
ddH₂O	Bring to 100 mL

Store at 4°C protected from light after preparation.

4) 10% APS

Preparation (10 mL):

① Weigh 1.00 g ammonium persulfate (APS) into a 15 mL centrifuge tube;

② Add double-distilled water to ~8–9 mL, vortex to dissolve, then bring to 10.0 mL;

③ Prepare fresh for immediate use or store at 4°C protected from light (≤1 week). For longer storage, aliquot 0.5–1.0 mL per tube, store at −20°C protected from light, and avoid repeated freeze–thaw; it is recommended to verify polymerization performance with a small trial before use.

Notes:

① Solid ammonium persulfate (APS) is highly hygroscopic, deliquesces, and loses activity. Purchase in small packages or aliquot immediately after receipt; store sealed in a dry place, or at −20 °C with desiccant and protected from light.

② After opening a small package, prepare a single batch of stock solution, then aliquot 1.0 mL per tube into 1.5 mL microcentrifuge tubes and freeze at −20 °C. Thaw one tube per use and avoid repeated freeze–thaw cycles or returning leftovers to the stock.

③ Storage and handling notes: Keep containers dry and clean; minimize exposure time after opening. Avoid contamination with metal ions as well as high temperature and strong light. Prepare stock solutions in small batches for near-term use; for long-term storage, remove particulates by membrane filtration before freezing.

④ Quality control: If caking, discoloration, or sluggish initiation of polymerization is observed after solution preparation, regard the APS as activity-degraded and discard; prepare a fresh batch.

5) TEMED

6) 4× Peptide gel buffer (3 M Tris-HCl; 0.4% SDS, pH 8.45)

Component	Amount
Tris	182 g
SDS	2.0 g (20 mL of 10% SDS)
HCl	Adjust to pH 8.45 with 1 mol/L
ddH₂O	Bring to 500 mL

Store at 4°C after preparation.

7) 10× Tris-tricine-SDS running buffer (dilute to 1× before use)

Prepare 10× TTS as per the table below (bring to 1000 mL), store at 4°C. For use, mix 50 mL 10× TTS with 450 mL ultrapure water to make 1× TTS for electrophoresis.

Component	Final Conc.	Amount
Tris	1 M	121.14 g
Tricine	1 M	179.2 g
SDS	1%	10 g
ddH₂O	Bring to 1000 mL

8) 1.0 mol/L Tris-HCl (pH 6.8) protein sample buffer (100 mL)

Preparation (100 mL):

Weigh 12.11 g Tris powder into a 100 mL beaker, add 80 mL distilled water, stir until fully dissolved, adjust to pH 6.8 with 1 mol/L HCl, transfer into a 100 mL volumetric flask, bring to 100 mL with distilled water, and store at 4°C.

9) 2× Tricine protein sample loading buffer (10 mL)

Component	Final Conc.	Amount
Tris-HCl (1 M, pH 6.8)	100 mM	1 mL
Glycerol	24%	2.4 mL
SDS	8%	0.8 g
DTT	0.2 M	0.31 g
Coomassie Brilliant Blue G-250	0.02%	2 mg
ddH₂O	Bring to 10 mL

10) Staining solution

Component	Amount
Glacial acetic acid	50 mL
Methanol	250 mL
Coomassie Brilliant Blue G-250	0.25 g
ddH₂O	Bring to 500 mL

11) Destaining solution

Component	Amount
Methanol	200 mL
Glacial acetic acid	70 mL
ddH₂O	Bring to 500 mL

12) 18% Tris-Tricine gel formulations

Separation gel（T=18% C=5%）	5 mL	8 mL	10 mL	16 mL	25 mL	32 mL
Ethylene glycol	1.50	2.40	3.00	4.80	7.50	9.60
40% Acr-Bis (19:1)	2.25	3.60	4.50	7.20	11.25	14.40
Gel buffer	1.25	2.00	2.50	4.00	6.25	8.00
10% APS	0.038	0.060	0.075	0.120	0.188	0.240
TEMED	0.004	0.006	0.008	0.012	0.019	0.024

Stacking gel（T=5% C=3.3%）	1 mL	2 mL	3 mL	4 mL	5 mL	6 mL
ddH₂O	0.612	1.22	1.83	2.45	3.06	3.67
40% Acr-Bis (29:1)	0.125	0.25	0.38	0.50	0.63	0.75
Gel buffer	0.250	0.50	0.75	1.00	1.25	1.50
10% APS	0.013	0.025	0.038	0.050	0.063	0.075
TEMED	0.001	0.002	0.003	0.004	0.005	0.006

3. Preparation of Polyacrylamide Gels (18%T, 5%C / 6 mL separation gel; 5%T, 3.3%C / 4 mL stacking gel)

(1) Preparation of gel casting mold

① Select glass plate thickness according to experimental objectives; for electrophoretic observation and transfer of small proteins, thinner formats (e.g., 1.0 mm, 0.75 mm) are preferred.

② Wipe evenly with 75% (v/v) ethanol to degrease, then rinse with distilled water; dry with lint-free wipes to remove particles and fingerprints.

③ Assemble the thick plate (spacer plate) and thin plate (short plate) into the casting stand per the manual, ensuring proper sealing and uniform clamping.

(2) Preparation of separation gel

① Using a 1 mL pipette, slowly dispense the separation gel solution along the inner side of the glass plates; stop when the liquid level is ~1.5 cm below the top of the front plate or ~0.5 cm below the comb teeth.

② Overlay ~1 cm of distilled water on the separation gel surface to level the interface and exclude air.

③ Let stand at room temperature for 30–60 min; polymerization is complete when a clear interface forms between the separation gel and overlay water. Discard the top water, tilt the plates, and wick away residual liquid with filter paper.

④ Prepare the stacking gel and slowly pour along the glass wall to near the top; insert the comb vertically to form wells and allow 30–60 min for polymerization.

⑤ After solidification, mount the gel with the plates into the electrophoresis tank; after adding running buffer, carefully remove the comb and prepare to load samples in sequence. For temporary storage, seal the gel with plates in a zip-lock bag at 4°C (a small amount of distilled water may be added to maintain humidity).

4. Protein Electrophoresis

(1) Sample preparation

① Preheat a water bath or dry block to 70–100°C.

② Transfer the required amount of protein samples into 1.5 mL microcentrifuge tubes, label sequentially, and record.

③ Add loading buffer at the specified ratio and mix gently to homogenize.

④ Heat in the water bath/dry block for 10–15 min.

⑤ Remove and cool to room temperature, briefly centrifuge for 10–30 s to collect liquid.

⑥ Arrange samples by number for loading.

(2) Electrophoresis

① Mount the gel with wells facing upward into the electrophoresis tank; add running buffer to the inner and outer chambers: the outer buffer level should cover the electrodes/platinum wire, and the inner buffer should fully submerge the wells.

② Remove the comb vertically and slowly; before loading, use a pipette to gently aspirate and dispense a small amount of buffer in each well to remove residual gel and bubbles.

③ Load samples with a micropipette according to the predefined order and record.

④ Set a constant voltage of 150 V; initial current ~45–55 mA, final current ~10–15 mA, run for 2–3 h; stop when the bromophenol blue front reaches the lower edge of the separation gel.

Operational Notes:

① Prefer using gel-loading pipette tips to ensure complete and accurate delivery of samples into the wells.

② Control the loading volume to avoid overloading or spilling into adjacent wells, which can cause band interference.

③ Do not insert the pipette tip too deeply — avoid touching the bottom of the well to prevent band distortion; likewise, avoid staying too high above the well to prevent sample diffusion into the buffer.

④ Complete sample loading continuously to minimize the residence time of samples in the wells and reduce the risk of diffusion.

(3) Staining

① Place the finished PAGE gel into a clean plastic container.

② Add staining solution to fully cover the gel and pre-soak for 1–2 min.

③ Gently shake on a rocker at room temperature for 10–15 min, avoiding overlap of gel pieces.

④ Remove and inspect color development.

References

1.Schägger H. Tricine–SDS-PAGE. Nature Protocols. 2006;1:16–22. doi:10.1038/nprot.2006.4.

2.Cao Z. [Tricine-SDS-PAGE method for effective separation of 1 kDa small peptides]. China Biotechnology. 2004;24(1):74–76. Chinese.

Aladdin: https://www.aladdinsci.com/