Tricine-SDS-PAGE

Syed R. Haider, Helen J. Reid and Barry L. Sharp

S.Haider@lboro.ac.uk; H.J.Reid@lboro.ac.uk; B.L.Sharp@lboro.ac.uk

Summary

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-

PAGE) is an efficient way of separating low molecular mass proteins. However, the

standard system is quite complicated and specifically may not be useful when the

separated proteins require to be recovered from the gel for quantitative analysis. Here

we describe a simplified system whereby these smaller proteins can be resolved in

comparatively low percentage gels which have high compatibility with modern

detectors such as UV and inductively coupled plasma-mass spectrometry (ICP-MS).

Keywords: Tricine-SDS-PAGE, Low molecular mass proteins, Running buffer, Gel

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1. Introduction

The separation of low molecular mass proteins (1-30kDa), or fragmented peptides

derived from enzymatic digestion, is a difficult task. To achieve such separations with

high resolution, slab SDS-PAGE based methods are frequently used and allow several

lanes to be run on a single gel under identical conditions. Thus it is easy to compare

and to determine the molecular masses of the separated species by calculating the

relative mobility of each band relative to standards run on the same gel The Laemmli-

SDS-PAGE method (1) is widely used for the separation of the proteins, however, for

the separation of low mass proteins this system requires a very high percentage gel

(>16%) or a gradient gel (2-3). Such gels have several disadvantages including

difficulties in casting, irreproducibility, fragility and short shelf life (4). Further, it is

difficult to quantitatively recover proteins in blotting and electroelution. The high

background signals of sulphur and phosphorus in high percentage gel also make them

unsuitable for laser ablation (LA)-ICP-MS detection of proteins (5-7).

Tricine-SDS-PAGE is a remarkably simple and efficient alternative way of separating

low molecular mass proteins/peptides in a single polyacrylamide gel with high

resolution. Usually, a 10% gel (with pH 8.45) is employed to separate proteins in the

range of 1-100 kDa using two running buffers: a cathode buffer, e.g. 100 mM Tris,

100 mM tricine and 0.1% (w/v) SDS, pH 8.25, and an anode buffer, e.g.100 mM Tris,

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pH 8.9. However, to obtain highly resolved bands, this method requires three gels

(stacking, spacer and resolving gel) (8-10) and addition of urea (4) in the resolving

gel. The use of three gels is tedious, potentially troublesome and always requires a

freshly prepared gel mixture (10). Addition of urea may also create problems in amino

acid sequencing (11), and although it is useful for the analysis of low mass proteins, it

crystallizes at low temperature and sometimes decomposes during sample preparation

(4). Recently, we have described an improved form of the tricine-SDS-PAGE method

employing a simplified procedure which does not require two different electrode

buffers, a spacer gel or addition of urea (12). This modified system is well suited for

quantitative analysis and shows excellent compatibility with detectors such as ICP-

MS or UV. For such analysis (and where the retention of metals is desired), the softest

conditions are preferred, that is low percentage gels and low concentration buffers.

For comparative purposes, the following provides descriptions of separations based

on the modified method reagent system and the original method reagent system.

These show that using the simplified approach of the modified system a different

buffer and pH are required and that the original reagent system does not work

efficiently under these conditions. The user does not need to follow the steps for the

original method, they are included only for information.

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2. Materials

Prepare all the solutions in ultra-pure water (18 M-Ω, e.g. from a Milli-Q water

purification system, Millipore Corporation, Bedford, MA). Store at room

temperature (if required), unless otherwise stated.

2.1 Tricine-SDS-PAGE Components

1. 2.5 M Tris buffer, pH 8.8 for modified, and pH 8.45 for original system (see

Note 1-2): Weigh 302.85 g of Tris-base and transfer it to a 1-L graduated

cylinder or glass beaker. Dissolve the base by adding 600 mL of deionized

water and adjust the pH using HCl. Make up to the 1 L with water. This gel

buffer can be stored at 4-8°C.

2. Polyacrylamide gel solution: It is highly recommended to purchase the

readymade polymer solution from the suppliers instead of preparing it in the

laboratory because acrylamide is recognised as a neurotoxin and suspected

carcinogen. For this work a 30% (w/v) solution of acrylamide: bisacrylamide

29:1 (Sigma Aldrich, Poole, UK) was used. This polyacrylamide solution

should be stored at 4-8°C.

3. Running buffer for modified system: 25 mM Tris, 25 mM tricine, 0.05% (w/v)

SDS. Dissolve 3.03 g Tris-base, 4.5 g tricine and 0.5 g SDS in 1-L of

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 deionized water (see Note 3). There is no need to adjust the pH. Store at 4-

8°C. (see Note 4).

4. Running buffer for original system: 100 mM Tris, 100 mM Tricine 0.1% (w/v)

SDS. Weigh 12.1 g Tris-base, 17.9 g tricine and 1 g SDS and dissolve in 1-L

of deionized water (see Note 3-5). There is no need to adjust the pH. Store at

4-8°C (see Note 4).

5. Sample or loading buffer: 2 x sample buffer (Sigma Aldrich, Poole, UK)

containing 100 mM Tris-HCl (pH 6.8) (see Note 6), 1% (w/v) SDS, 4% (v/v)

2-mercaptoethanol, 0.02% (w/v) Coomassie Brilliant Blue (CBB) and 24%

(w/v) glycerol. Store at -20°C (see Note 4).

6. Ammonium persulphate (APS) solution: Weigh 0.03 g of APS and dissolve in

1-mL of deionized water (see Note 7).

7. N,N,N’,N’- tetramethylethylenediamine (TEMED) (Sigma Aldrich, Poole,

UK) (see Note 8).

8. Coomassie Brilliant Blue (CBB) staining solution: Dissolve 0.025-0.030

g of CBB in 100 mL of 10% (v/v) acetic acid solution (see Note 9).

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 9. Acetic acid (10% v/v) de-staining solution: Prepare 100 mL of 10%

(v/v) acetic solution (see Note 9)

10. Glutaraldehyde (5% v/v) fixing solution: Prepare 5% glutaraldehyde

solution by diluting 5 times the 25% (v/v) glutaraldehyde stock solution

(Sigma Aldrich, Poole, UK) (see Note 9). Both stock and diluted solutions

should be stored at -20°C (see Note 4).

2.2 Protein Mass Ladder

Protein mass ladder: (a) with a mass range of 2.5-200 kDa (Invitrogen

Limited, Paisley, UK) and (b) 1.0-26.6 kDa Ultra Low Molecular Weight

MarkerTM (Sigma Aldrich, Poole, UK). Store the protein mass ladder

(a) at 4-8 ºC and protein mass ladder (b) at -20 ºC (see Note 4). For the detail

of the protein mass ladders (a) and (b), see Table 1.

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2.3 Protein Digestion

1. 1, 4-dithio-DL-threitol (DTT) (100 mM): Weigh 0.015 g and dissolve in 1 mL

of deionized water. Store at 4-8°C.

2. Iodoacetamide (IAA) (100 mM): Weigh 0.02 g and dissolve in 1 mL of

deionized water. Store at 4-8°C.

3. Ammonium bicarbonate (NH4HCO3) (50 mM): Dissolve 0.04 g of

NH4HCO3 in 10 mL of deionized water (see Note 10).

4. Sequencing grade modified trypsin (0.1 µg/µL) (Promega, Delta

House, Southampton, UK): Dissolve 100 µg of the lyophilized powder

of modified trypsin in 1 mL of deionized water (see Note 11). Store trypsin

solution in aliquots at -20°C. For long term storage use -80°C.

5. Sample for protein digestion was prepared by dissolving 1 mg of

β-casein in 1 mL of deionized water.

3. Methods

All the procedures should be carried out at room temperature.

3.1 Protein Digestion

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 Protein digestion in solution for a comparative study should be performed

according to the following method. Here β-casein is used as a model protein.

1. Add 10 µL of 1 mg/mL β-casein solution in the mixture of 15 µL of 50 mM

NH4HCO3 and 1.5 µL of 100 mM DTT solution and incubate at 90 ºC for 5-10

min.

2. After cooling to room temperature, add 3 µL of 100 mM IAA to this solution

and incubate for 25 minutes in the dark.

3. Add 1 µL of 0.1 µg/µL sequencing grade modified trypsin and incubate at 37

ºC for 4 hours.

4. Finally, add an additional 1 µL of trypsin and incubate overnight at 30 ºC.

Next day, reduce digest mixture to 10 µL using vacuum.

3.2 Gel Casting

1. Prepare all stacking (4% w/v) and resolving gels mixture for 7, 9 and 10%

(w/v) gels for both the original and modified methods as shown in Table 2

(see Note 12-14).

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2. Cast all the gels in gel cassettes at 7.3 cm height x 8 cm width and 0.75 mm

thickness using a mini- gel casting apparatus (Bio-Rad, Hemel Hempstead,

UK) (see Note 15-16).

3. Firstly, inject the resolving gel mixture into the gel cassette (see Note 17-18).

4. Do not fill the resolving gel mixture to the top of the gel plates. Leave a space

of approximately 1-2 cm from the top. Quickly fill this space with butanol or

70% ethanol (see Note 19-20).

5. The resolving gel usually takes 10-15 minutes for polymerization. Check the

polymerization of the gel in the centrifuge tube and if the gel has been

polymerized then pour off the butanol (or 70% ethanol) form the top of the

resolving gel. Start injecting the stacking gel mixture onto the polymerized

resolving gel in the gel cassette and fill up to the top of the short plates and

then quickly insert the gel combs for the loading wells. Check the

polymerization of the stacking gel in the centrifuge tube, or by just slightly

lifting the combs, and if the stacking gel is polymerized then remove the gel

cassette sandwiche from gel casting frame and set this into the electrode

assembly. The short plates must be pointing inward in the dual plate electrode

assembly (see Fig. 1).

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3.3 Sample Preparation

Mix 10 µL of the sample buffer with 10 µL of the digest mixture. Dilute 20-

fold the protein mass ladder (b) with the 1 x sample buffer and heat at 65 ºC

(see Note 21). There is no need to dilute or heat the protein mass ladder (a).

3.4 Sample Loading and Gel Running

1. Place the electrode assembly containing the 2 gel cassettes in the running

buffer tank.

2. Fill the tank with the running buffer and gently remove the combs. Wash the

loading wells with the running buffer using 1 mL pipette tips to remove any

un-polymerized acrylamide.

3. Load 7 µL of protein mass ladder (a), 5 µL of protein mass ladder (b), and 10

µL of β-casein digest (as prepared above) in each well of the 7, 9 and 10%

(w/v) slab gels for the modified and original tricine-SDS-PAGE methods.

4. Apply a constant voltage of 125-150 V. Do not stop until the dye front touches

the bottom (see Note 22-23).

3.5 Gel Fixing, Staining and De-staining

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 1. Fix the gels for 25 minutes (using a shaker) with the fixing solution of 5%

(v/v) glutaraldehyde.

2. Stain each gel for 20 minutes (using a shaker) using CBB staining solution.

3. Perform de-staining for 20 minutes (using a shaker) using 10% (v/v) acetic

acid solution (see Note 24-25). See Fig. 2, 3 and 4 for typical results.

4. Notes

1. The “original method” refers to the previously described (8-9) tricine-SDS

PAGE and “modified method” (12) refers to the modification here described.

2. The previously described methods for tricine-SDS-PAGE employed 3.0 M

tris-base whilst the modified method uses 2.5 M so here, for the comparative

study the concentration of the gel buffer was kept to 2.5 M for both systems.

3. The running buffer can be prepared at 10 x concentration and can be diluted

accordingly.

4. All the solutions should be used at room temperature.

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5. For comparative study between the modified and original tricine SDS-PAGE

system, only one running buffer was used.

6. Tris-base with pH of 7.0 can also be used instead of pH 6.8.

7. APS must always be freshly prepared.

8. TEMED is a very odourous neurotoxin so extra care is required when handling

it.

9. Avoid using methanol in staining, de-staining and fixing solutions as

this may cause loss of the low molecular mass proteins.

10. There is no need to adjust the pH of the ammonium bicarbonate solution.

11. Sequencing grade modified trypsin does not suffer from self autolysis and

minimizes miss-cleavages during the digestion.

12. We prefer a 10-50 mL centrifuge tube for preparing the gel mixture.

13. Do not include SDS, glycerol or urea in any of the gels.

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14. TEMED and APS must be added last in the gel mixture which requires

quick injection into the gel cassettes, otherwise gel will be polymerized in the

tube.

15. Acrylamide is recognized as a neurotoxin, so gloves must be worn all

the time and the work must be done in a properly ventilated area.

16. For the purpose of comparing the original and modified methods the same

gel casting protocol was used for the both systems.

17. Inject the gel mixture slowly into the gel cassettes using 5 mL pipette tips and

avoid bubble formation which may increase the electrical

resistance between the electrodes during electrophoresis.

18. In case of direct contact of the gel mixture with the skin wash the skin

thoroughly with water and seek medical advice if the contact

is severe because acrylamide can readily be absorbed by the skin.

19. Butanol or 70% ethanol provides a smooth resolving gel surface.

20. The butanol or 70% ethanol must be removed before casting the stacking gel

on the top of the resolving gel.

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 21. For example, Sigma Aldrich (Poole, UK) provides 10 mL of 2x sample buffer

free with the protein mass ladder (b).

22. Do not overfill the wells otherwise they will contaminate the neighbouring

wells.

23. For rapid separation, a higher voltage of 200-250 can be applied, however, it is

not preferred because it increases the temperature of the buffer which may

cause a significant change in the pH of the running buffer and/or resolving gel.

24. Wash the gel with deionized water after each of the fixing, staining and de-

staining steps.

25. For best results, do not reuse fixing, staining and de-staining solutions.

26. Confirmation of the phosphopeptide bands were obtained by whole gel

elution and ICP-MS detection of 31P (for detail, see reference 12).

Acknowledgments

This work was supported by a grant from Loughborough University, Loughborough,

Leicestershire, LE11 3TU, UK.

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