Protein Tris-Acetate Electrophoresis System Standard Operating Procedure

1. Experimental Equipment

ØVertical Electrophoresis System: Power supply, electrophoresis tank, and associated accessories

ØGel Casting Assembly: Thick glass plate (spacer plate, 0.75/1.0/1.5 mm), thin glass plate (short plate), and plastic frame

ØPipetting Tools: Micropipettes and compatible tips (1 mL, 200 μL, 10 μL)

ØSampling and Storage Consumables: Centrifuge tubes (1.5 mL, 15 mL, 50 mL)

ØGlassware: Beaker, volumetric flask (100 mL), graduated cylinders (100 mL, 500 mL, 1000 mL), glass rods

ØReagent Containers: Wide-mouth reagent bottles (500 mL, 1000 mL), amber bottles (250 mL, 500 mL)

ØLaboratory Consumables: Filter paper, lint-free wipes, plastic wrap/sealing bags

ØMeasurement and Monitoring: pH meter, etc.

2. Experimental Reagents

Ø40% Acrylamide Solution: N,N′-Methylenebisacrylamide 37.5:1 mixture (40% Acr-Bis)

Component	Amount
Acrylamide (MW: 71.08)	38.96 g
Bisacrylamide (MW: 154.17)	1.04 g

Preparation (100 mL):

①Weigh 38.96 g acrylamide (Acr) and 1.04 g N,N′-methylenebisacrylamide (Bis) into a 100 mL beaker.

② Add 50 mL distilled water (warm water aids dissolution) and stir magnetically until completely dissolved.

③ Transfer to a 100 mL volumetric flask and bring to volume with distilled water. Mix gently to homogenize.

Notes:

① Storage and Stability: Acrylamide can slowly undergo deamination under light exposure and alkaline conditions, forming acrylic acid/diacrylic acid. After preparation, verify that the solution pH is ≤ 7.0 and store at 4 °C protected from light; a recommended shelf life is ≤ 2 months. If insoluble material appears (from reagent impurities or precipitation during storage), filter through paper and then aliquot for storage.

② Polymerization and Gelation Mechanism: Acrylamide monomers polymerize into long chains via free-radical initiation; in the presence of N,N′-methylenebisacrylamide (Bis), crosslinking occurs to form a three-dimensional gel network. The gel pore size is jointly determined by chain length and crosslinking density, thereby influencing the separation range and resolution.

Ø3 mol/L Tris–Acetate Buffer (pH 7.0)

Preparation (100 mL):

① Weigh 36.34 g Tris into a 100 mL beaker.

② Add 80 mL distilled water and stir magnetically until dissolved.

③ Adjust to pH 7.0 dropwise using glacial acetic acid while monitoring pH.

④ Transfer to a 100 mL volumetric flask and make up to volume with distilled water. Store at 4°C.

Ø10% Sodium Dodecyl Sulfate (SDS) Solution

Ø10% Ammonium Persulfate (APS) Solution

Preparation (10 mL):

① Weigh 1.00 g 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 total volume.

③ Aliquot 1.0 mL per 1.5 mL microtube, store protected from light at –20°C.

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.

ØTetramethylethylenediamine (TEMED)

ØElectrophoresis Buffer (10× Tris–Acetate SDS Running Buffer, 10× TAS)

Component	Amount	Final 1× Concentration
Tris (MW: 121.14)	60.57 g	50 mM
Sodium acetate (MW: 82.03)	41.02 g	50 mM
SDS (MW: 288.38)	10 g	0.1%
Sodium bisulfite (MW: 104.1)	2.5 g	2.4 mM
ddH₂O	Bring to 1000 mL

pH=8.24

Preparation (1000 mL):

① Weigh 60.57 g Tris, 41.02 g sodium acetate (anhydrous), 10.00 g SDS, and 2.50 g sodium bisulfite into a 1000 mL beaker.

② Add 800 mL distilled water and stir magnetically until completely dissolved.

③ Transfer to a 1000 mL reagent bottle (or via a graduated cylinder), and bring to 1000.0 mL with distilled water.

④ Store at room temperature sealed; recommended shelf life: 1 month.

⑤ Dilute 10× stock 1:10 with distilled water to obtain 1× working buffer. pH should be around 8.24; if deviation >0.5, adjust and check water pH, reagent purity, and storage conditions.

ØTransfer Buffer (10× Tris–Acetate Transferring Buffer)

Component	Amount	Final 1× Concentration
Tris (MW: 121.14)	30.29 g	25 mM
Sodium acetate (anhydrous, MW: 82.03)	20.51 g	25 mM
EDTA (0.5 M EDTA·2Na, pH 7.8–8.2)	20 mL	1 mM
Sodium bisulfite (MW: 104.1)	1.35 g	1.3 mM
ddH₂O	Bring to 1000 mL

pH 7.2

Preparation (1000 mL):

① Weigh 30.29 g Tris, 20.51 g sodium acetate, and 1.35 g sodium bisulfite into a 1000 mL beaker.

② Add 20 mL 0.5 M EDTA and 800 mL distilled water; stir until fully dissolved.

③ Transfer to a 1000 mL reagent bottle, bring to 1000.0 mL with distilled water, and store at 4°C (stable for 1 month).

④ For use, add 20% (v/v) methanol and dilute to 1× working concentration with distilled water.

ØSeparation and Stacking Gel Formulation Tables

Separation Gel（T=7%）

Component	5 mL	8 mL	10 mL	16 mL	20 mL	25 mL
ddH₂O (mL)	3.76	6.01	7.52	12.02	15.03	18.79
40% Acr–Bis (mL)	0.88	1.40	1.75	2.80	3.50	4.38
3 M Tris–Acetate pH 7.0 (mL)	0.34	0.54	0.68	1.08	1.35	1.69
10% APS (µL)	24	38	48	77	96	120
TEMED (µL)	6	10	12	19	24	30

Separation Gel（T=6%）

Component	5 mL	8 mL	10 mL	16 mL	20 mL	25 mL
ddH₂O (mL)	3.91	6.26	7.82	12.52	15.65	19.56
40% Acr–Bis (mL)	0.75	1.20	1.50	2.40	3.00	3.75
3 M Tris–Acetate pH 7.0 (mL)	0.34	0.54	0.68	1.08	1.35	1.69
10% APS (µL)	24	38	48	77	96	120
TEMED (µL)	6	10	12	19	24	30

Separation Gel（T=5%）

Component	5 mL	8 mL	10 mL	16 mL	20 mL	25 mL
ddH₂O (mL)	4.04	6.46	8.07	13.00	16.15	20.19
40% Acr–Bis (mL)	0.62	1.00	1.25	2.00	2.50	3.12
3 M Tris–Acetate pH 7.0 (mL)	0.34	0.54	0.68	1.08	1.35	1.69
10% APS (µL)	24	38	48	77	96	120
TEMED (µL)	6	10	12	19	24	30

Separation Gel（T=4%）

Component	5 mL	8 mL	10 mL	16 mL	20 mL	25 mL
ddH₂O (mL)	4.16	6.66	8.32	13.32	16.65	20.81
40% Acr–Bis (mL)	0.50	0.80	1.00	1.60	2.00	2.50
3 M Tris–Acetate pH 7.0 (mL)	0.34	0.54	0.68	1.08	1.35	1.69
10% APS (µL)	24	38	48	77	96	120
TEMED (µL)	6	10	12	19	24	30

Loading Gel（3%）

Component	1 mL	2 mL	3 mL	4 mL	5 mL	6 mL
ddH₂O (mL)	0.85	1.70	2.55	3.41	4.26	5.11
40% Acr–Bis (mL)	0.08	0.15	0.23	0.30	0.38	0.45
3 M Tris–Acetate pH 7.0 (mL)	0.07	0.14	0.20	0.27	0.34	0.41
10% APS (µL)	5	10	15	20	25	30
TEMED (µL)	1	2	3	4	5	6

Note:Values exclude APS/TEMED; add freshly before use.

ØNeutral Sample Buffer (4× LDS Loading Buffer)

Component	Amount (for 10 mL 4×)	Final 4× Concentration
Tris (MW: 121.14)	1.21 g	1 M
LDShttps://www.aladdinsci.com/catalogsearch/result/?q=2044-56-6 (Lithium dodecyl sulfate)	0.80 g	8% (w/v)
EDTA-2Na-2H₂O (MW: 372.24)	3.72 mg	1 mM
Glycerol	4.0–5.0 mL	40–50% (v/v)
Coomassie Brilliant Blue G-250	7.5 mg	0.075% (w/v)
Phenol Red	2.5 mg	0.025% (w/v)
ddH₂O	Bring to 10.0 mL

pH 8.5

Preparation (10 mL):

① Add 1.21 g Tris, 0.8 g LDS, 7.5 mg Coomassie Brilliant Blue, and 2.5 mg phenol red into a 10 mL centrifuge tube.

② Add 5 mL glycerol and ~2 mL distilled water; mix until dissolved.

③ Bring to 10.0 mL with distilled water and mix well. Store at 4°C (stable for 1 month).

④ For reducing samples, add DTT to a final concentration of 100 mM in the 1× working solution.

ØAbsolute Ethanol

ØWater Systems: Distilled water (dH₂O) or reverse osmosis (RO) water, double-distilled water (ddH₂O), and ultrapure water (UP).

3. Gel Preparation

(1) Preparation of Gel Casting Mold

① Select glass plates of appropriate thickness according to experimental needs (matching spacer specifications).

② Wipe with 75% (v/v) ethanol to degrease, rinse with distilled water, and dry using lint-free tissue.

③ Assemble and fix in the casting stand per manufacturer’s instructions, ensuring even clamping and proper sealing.

(2) Casting of Separation Gel

① Add distilled water, 40% Acr–Bis, and 3 mol/L Tris–Acetate (pH 7.0) in sequence and mix gently.

② Add freshly prepared 10% APS and TEMED, mix quickly and uniformly.

③ Pour slowly along the glass plate wall until the liquid level is ~1.5 cm below the top of the short plate (or ~0.5 cm below comb teeth).

④ Overlay ~1 cm distilled water on the surface to level and exclude air.

⑤ Allow to polymerize for 20–40 min at room temperature; when a clear interface appears, discard the top water and blot residual liquid with filter paper.

(3) Casting of Stacking Gel

① Add distilled water, 40% Acr–Bis, and 3 mol/L Tris–Acetate (pH 7.0) sequentially and mix thoroughly.

② Add freshly prepared 10% APS and TEMED, mix immediately and evenly.

③ Pour slowly along the glass wall up to near the top; insert the comb vertically to form wells and let polymerize for 10–20 min.

④ After polymerization, assemble the gel with the plates into the electrophoresis tank; fill with running buffer, gently remove the comb, and proceed to sample loading.

If temporary storage is needed, 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) Operational Notes

① When preparing mini gels, 15 mL or 50 mL centrifuge tubes can be used as containers. Mix thoroughly by gentle rotation or repeated pipetting.

② For large-volume preparation, it is recommended to use a beaker of matching capacity and stir thoroughly with a glass rod.

③ Regardless of the method used, avoid bubble formation throughout the process; if small bubbles appear, allow them to rise slowly along the wall and escape before casting the gel.

4. Protein Electrophoresis

(1) Sample Preparation

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

② Transfer required protein samples to 1.5 mL microtubes and label sequentially.

③ Add loading buffer in the specified ratio (choose reducing or non-reducing neutral formulation as required) and mix gently.

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

⑤ Cool to room temperature, briefly centrifuge 10–30 s to collect liquid.

⑥ Arrange samples in order for loading.

(2) Electrophoresis

① Mount the gel (wells upward) into the electrophoresis tank. Add running buffer to both inner and outer chambers: the outer buffer should cover the electrodes/platinum wire, and the inner buffer should fully submerge the wells.

② Slowly remove the comb vertically. Before loading, pipette a small volume of running buffer into each well to remove residual gel and bubbles.

③ Load samples using a micropipette according to sequence and record the order.

④ Run at 130 V (I ≈ 115 mA) for approximately 1 h 15 min. Stop when the tracking dye front reaches the bottom. Adjust duration as needed based on target protein size and resolution.

(3) 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.

References

1.Methods in molecular biology, 869, 205–213.  

2.Cubillos-Rojas M, et al. Tris-acetate polyacrylamide gradient gels for the simultaneous electrophoretic analysis of proteins of very high and low molecular mass. Methods in molecular biology, 869, 205–213. https://doi.org/10.1007/978-1-61779-821-4_17

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