Standard Operating Procedure (SOP) for Bis-Tris Polyacrylamide Gel Electrophoresis (SDS-PAGE)

1. Experimental Apparatus

ØMeasuring and glassware: Graduated cylinders (100 mL, 500 mL, 1000 mL), volumetric flasks (100 mL), beakers, glass rods.

ØStorage and sampling containers: Reagent bottles (500 mL, 1000 mL), amber bottles (250 mL, 500 mL), centrifuge tubes (1.5 mL, 15 mL, 50 mL), amber centrifuge tubes (for aliquoting TEMED), plastic wrap/sealing bags.

ØElectrophoresis and gel-casting components: Vertical electrophoresis system (including power supply and tank), gel-casting glass plates (spacer plates of 0.75/1.0/1.5 mm thickness, short plates), and supporting frames.

ØPipetting tools: Micropipettes and compatible tips (10 μL, 200 μL, 1 mL, 5 mL).

ØFiltration and cleaning materials: Filter paper, lint-free wipes.

ØMeasuring/instrumentation: pH meter, analytical balance, vortex mixer.

2. Experimental Reagents

Ø32% Acrylamide/Bis-acrylamide Solution (Acr-Bis)

Component	Amount	Final Concentration
Acrylamide (MW: 71.08)	30 g	30%
N,N′-Methylenebisacrylamide (MW: 154.17)	2 g	2%
ddH₂O	To 100 mL

Preparation (100 mL):

①Weigh 30.00 g of acrylamide and 2.00 g of N,N′-methylenebisacrylamide into a 100 mL beaker.

②Add 50 mL ultrapure water and stir magnetically until fully dissolved.

③Transfer the solution into a 100 mL graduated cylinder and make up to 100.0 mL with ultrapure water; mix gently to homogenize.

④Filter through paper (or 0.45 μm membrane), transfer to an amber bottle, store at 4°C in the dark. Recommended shelf life: 1–2 months.

Ø3.5× Bis-Tris Gel Buffer

Component	Quantity	Final Concentration (1×)
Bis-Tris (MW: 209.24)	2.616 g	35 mM
ddH₂O	To 100 mL

Preparation (100 mL):

①Weigh 2.616 g Bis-Tris into a 100 mL beaker.

②Add 50 mL deionized water and stir magnetically until fully dissolved.

③Adjust pH to 6.5–6.8 dropwise with concentrated HCl while monitoring pH.

④Transfer into a 100 mL graduated cylinder, make up to 100.0 mL with deionized water, filter, aliquot, seal, and store at 4°C.

Ø10% Ammonium Persulfate (APS) and Tetramethylethylenediamine (TEMED)

ØElectrophoresis Buffers (20× MES Running Buffer, 20× MOPS Running Buffer)

(1) 20× MES Running Buffer (for proteins <20 kDa)

Component	Quantity	Final Concentration (1×)
MES·H₂O (MW: 213.25)	213 g	50 mM
Tris (MW: 121.14)	121.14 g	50 mM
EDTA·2Na·2H₂O (MW: 372.24)	7.44 g	1 mM
SDS (MW: 288.38)	20 g	0.1%
ddH₂O	To 1000 mL

pH7.3

Preparation (1000 mL):

①Weigh and add sequentially: MES·H₂O 213.0 g, Tris 121.14 g, EDTA·2Na·2H₂O 7.44 g, and SDS 20.00 g into a 1000 mL beaker.

②Add 600 mL deionized water and stir until fully dissolved.

③Transfer to a 1000 mL graduated cylinder and bring to volume with deionized water. pH adjustment not required.

④Aliquot, seal, and store at 4°C.

(2) 20× MOPS Running Buffer (for proteins >20 kDa)

Component	Quantity	Final Concentration (1×)
MOPS (MW: 209.26)	209.26 g	50 mM
Tris (MW: 121.14)	121.14 g	50 mM
EDTA·2Na·2H₂O (MW: 372.24)	7.44 g	1 mM
SDS (MW: 288.38)	20 g	0.1%
ddH₂O	To 1000 mL

pH7.7

Preparation (1000 mL):

①Weigh and add into a 1000 mL beaker: MOPS 209.26 g, Tris 121.14 g, EDTA·2Na·2H₂O 7.44 g, and SDS 20.00 g.

②Add 600 mL deionized water, stir until dissolved.

③Transfer to a 1000 mL graduated cylinder, make up to volume with deionized water. pH adjustment not required.

④Aliquot, seal, and store at 4°C.

(3) Gel Composition Tables (1.5 mm thick gels)

	8%					12%
Resolving Gel	1gel	2gels	4gels	6gels	8gels	1gel	2gels	4gels
ddH₂O	3.7 ml	7.4 ml	14.8 ml	22.2 ml	29.6 ml	2.7 ml	5.4 ml	10.8 ml
3.5× Bis-Tris buffer	2.3 ml	4.6 ml	9.2 ml	13.8 ml	18.4 ml	2.3 ml	4.6 ml	9.2 ml
32% Acrylamide stock	2.0 ml	4.0 ml	8.0 ml	12 ml	16 ml	3.0 ml	4.0 ml	8.0 ml
10% APS	75 µl	150 µl	300 µl	450 µl	600 µl	50 µl	100 µl	200 µl
TEMED	5 µl	10 µl	20 µl	30 µl	40 µl	3 µl	6 µl	12 µl
Total Volume	8 ml	16 ml	32 ml	48 ml	64 ml	8 ml	16 ml	32 ml

Stacking Gel	1gel	2gels	4gels	6gels	8gels
ddH₂O	1.76 ml	3.52 ml	7.04 ml	10.56 ml	14.08 ml
3.5× Bis-Tris buffer	0.86 ml	1.72 ml	3.44 ml	5.16 ml	6.88 ml
32% Acrylamide stock	0.38 ml	0.76 ml	1.52 ml	2.28 ml	3.04 ml
10% APS	20 µl	40 µl	80 µl	120 µl	160 µl
TEMED	4 µl	8 µl	16 µl	24 µl	32 µl
Total Volume	3 ml	6 ml	12 ml	18 ml	24 ml

Note: Total volume refers to main components; APS and TEMED are added separately and not included in the total.

Ø4× LDS Loading Buffer

Component	Quantity	Final Concentration (1×)
Tris·HCl (MW: 157.20)	666 mg	106 mM
Tris (MW: 121.14)	683 mg	141 mM
LDS (MW: 272.33)	800 mg	2% (w/v)
EDTA·2Na·2H₂O (MW: 372.24)	7.59 mg	0.51 mM
Glycerol (MW: 92.09)	4.0 g	10% (w/v)
Coomassie Brilliant Blue G-250 (MW: 854.04)	7.52 mg	0.22 mM
Phenol Red (MW: 354.38)	2.48 mg	0.175 mM
ddH₂O	To 10 mL

Preparation (10 mL):

①Add sequentially: Tris·HCl 0.666 g, Tris 0.683 g, LDS 0.800 g, EDTA·2Na·2H₂O 7.59 mg, and glycerol 4.000 g.

②Add 7.52 mg Coomassie Brilliant Blue G-250 and 2.48 mg Phenol Red; dissolve completely.

③Add deionized water to ~8–9 mL, dissolve fully, then make up to 10.0 mL; vortex to mix.

④Aliquot 1.0 mL per 1.5 mL microcentrifuge tube, store at 4°C; valid for 6 months.

ØddH₂O

Sample Preparation

Reagent	Reduced Sample	Non-reduced Sample
Sample	X μL	X μL
4× LDS Loading Buffer	2.5 μL	2.5 μL
Reducing agent (10×, 500 mM DTT or 25% β-ME)	1 μL	—
Deionized water	To make total volume 6.5 μL	To make total volume 7.5 μL
Total Volume	10 μL	10 μL

3. Polyacrylamide Gel Preparation

(1) Gel Mold Preparation

①Select glass plates of suitable thickness and size (with matching spacers).

②Wash thoroughly with neutral detergent to remove grease/residue, rinse with water, and air dry.

③Degrease evenly with 75% (v/v) ethanol, rinse again with distilled/deionized water, and dry completely in a dust-free environment.

④Before use, wipe contact surfaces with lint-free paper to remove fingerprints and particles.

⑤Assemble according to manufacturer instructions; check spacer orientation and seal integrity, ensure uniform clamping pressure, and perform a quick leak test with water before casting.

(2) Casting the Resolving Gel

①In a 50 mL centrifuge tube, sequentially add ultrapure water, 32% Acr-Bis solution, and 3.5× Bis-Tris buffer; mix gently until homogeneous.

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

③Using a 5 mL pipette, slowly pour the resolving gel solution along the inner wall of the glass plate; stop at ~1.5 cm below the top of the short plate (approximately 3.75 mL for a 6×8 cm, 1.5 mm gel).

④Overlay the gel surface with ~1 cm distilled water to level the interface and exclude air.

⑤Allow to polymerize for 20–40 min; once a clear interface forms between the gel and water, polymerization is complete. Discard the overlayer, tilt the plate, and remove residual water with filter paper.

(3) Casting the Stacking Gel

①In a 50 mL centrifuge tube, sequentially add ultrapure water, 32% Acr-Bis solution, and 3.5× Bis-Tris buffer; mix gently until homogeneous.

②Add freshly prepared 10% APS and TEMED; mix rapidly and thoroughly.

③Slowly pour the stacking gel solution along the inner plate wall up to near the top edge; insert the comb vertically to form wells. Allow to polymerize for 10–20 min at room temperature.

④Once polymerization is complete, use the gel within 30 min; if storage is required, seal the gel with glass plates in a self-sealing bag and store at 4°C (optionally adding a small amount of distilled water to maintain humidity).

⑤Before use, place the gel (with plates) into the electrophoresis tank, add running buffer, and gently remove the comb; prepare for loading.

(4) Practical Tips

①For mini gels, use 15 mL or 50 mL centrifuge tubes as mixing vessels; mix by gentle tube rotation or by pipetting up and down repeatedly.

②For larger volumes, use an appropriately sized beaker and mix thoroughly with a glass stir rod.

③Minimize bubbles regardless of the mixing method. If small bubbles form, allow them to rise along the wall and dissipate before casting the gel.

4. Protein Electrophoresis

(1) Sample Preparation

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

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

③Add the sample buffer in the specified ratio, mix gently until homogeneous.

④Heat the samples at 70–100°C for 5 min.

⑤Cool to room temperature and briefly centrifuge (10–30 s) to collect condensate.

⑥Arrange samples in order and prepare for loading.

(2) Electrophoresis Operation

①Mount the gel (wells facing up) into the electrophoresis cell; add running buffer to both inner and outer chambers — ensure the outer buffer covers the electrodes/platinum wire and the inner buffer fully submerges the wells.

②Carefully and slowly remove the comb vertically. Before loading, aspirate and flush each well several times with a small amount of buffer to remove residual gel or bubbles.

③Load samples with a micropipette in the designated order, recording sample sequence and volume.

④Set constant voltage at 120 V and begin electrophoresis. Stop when the phenol red dye front reaches the bottom of the gel. Adjust run time based on the molecular weight of target proteins.

(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.

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