Electrophoresis methods

Electrophoresis is the movement of positively or negatively charged molecules in an electric field. It is an analytical tool, indispensable across a broad range of the biosciences, particularly in analytical studies of proteins.

3.6.4.1 Tris-Tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Tris-Tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine–SDS-PAGE) ^[323] is commonly used to separate proteins in the mass range from 1 to 100 kDa because of Tricine, as the trailing ion, allows the resolution of proteins smaller than 30 kDa.

Tricine–SDS-PAGE is also preferentially used as a proteomic tool to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension.

SDS-PAGE was performed using the Mini-PROTEAN^® III gel electrophoresis system (BioRad, München, Germany). The dimensions of the gel are 90 x 60 x 1 mm. SDS-polyacrylamide gels were cast between two glass plates using a Bio-Rad mini-gel casting system. To obtain optimal resolution of proteins, a stacking gel was poured over the top of the separating gel. The stacking gel allows the proteins in a lane to be concentrated into a tight band before entering the separating gel and produces a gel with tighter or better separated protein bands. The size of the pores created in the gel is inversely related to the amount of acrylamide used, therefore lower percentage of acrylamide gels are better for resolving high molecular weight proteins, while much higher percentages are needed to resolve smaller

proteins. Table 17 and 18 provide compositions for stock solutions and preparing gels with different acrylamide concentrations.

Table 17. Stock solutions for Tris-Tricine SDS-PAGE

Buffer Tris (M) Tricine (M) pH SDS (%)

Anode buffer 0.2 - 8.9^a -

Cathode buffer 0.1 0.1 8.25^b 0.1

Tris-HCl/SDS buffer 3.0 - 8.45 ^a 0.3

a Adjusted with HCl

b No correction of the pH, which is around 8.25

Table 18. Composition of Tris-Tricine SDS-PAGE gel

-2 gels- Separating gel 10 % Separating gel 15 % Stacking gel

Acrylamide ^a 4.9 ml 7.5 ml 972 ml

Tris-HCl/SDS 5 ml 5 ml 1.86 ml

MilliQ 3.5 ml 0.9 ml 4.67 ml

Glycerol 1.58 ml 1.58 ml ---

APS ^b 75 ml 75 µl 40 µl

TEMED ^c 10 ml 10 µl 4.5 µl

a 30 % (w/v) Acrylamide, 0.8 % (w/v) N, N’- Methylenebisacrylamide

b 10 % Amonium peroxidsulphate;

c N’, N’, N’, N’-tetramethyl-ethylenediamine.

The sample was dissolved in a SDS reducing buffer (5 mM Tris-HCl, 4 % (w/v) SDS, 25 % (w/v) glycerol, 0.02 % (w/v) bromophenol blue, pH 6.8) and boiled for 5 min at 95 °C. Gel electrophoresis was carried out using a Power/PAC 1000 power supply (Bio-Rad, München, Germany) at a constant voltage of 60 V for ca. 15 min, until the tracking dye entered the separating gel, and at 120 V for ca. 1-2 h, until the tracking dye reached the anodic end of the separating gel. After separation in gels, proteins were visualized by silver staining or sensitive colloidal Coomassie Blue, and scanned using a GS-710 Calibrated Imaging Densitometer (Bio-Rad, München, Germany) with the Multi Analyst image analysis software (BioRad, Richmond, CA, USA) or electrophoresis gel imaging scanner analyzer (La Vision Bio Tec GmbH, Germany) to visualize proteins without staining. The molecular weights of unknown

proteins were estimated by running standard proteins of known ultra low range molecular weights in a separate lane of the same gel (Table 19).

Table 19. Ultra low molecular weight marker proteins for SDS-PAGE analysis. (M3546, Sigma, USA) Ultra low molecular weight marker proteins Molecular weight (Da) Triosephosphate Isomerase from rabbit muscle 26,600

Myoglobin from horse heart 17,000

-Lactalbumin from bovine milk 14,200

Aprotinin from bovine lung 6,500

Insulin Chain B, oxidized, bovine 3,496

Bradykinin 1,060

3.6.4.2 Sensitive colloidal coomassie staining

Coomassie Brilliant Blue G-250 is a convenient commonly used stain for visualizing proteins after electrophoretic separation. Proteins stained with Coomassie Brilliant Blue G-250 turn an intense blue color and are easily distinguished on polyacrylamide gels. The reagent is prepared under acidic conditions, causing the dye to assume a doubly protonated cationic structure. In this form, the dye is red brown. When it binds to proteins, it is converted to a stable anionic form, which is blue. The dye binds particularly to basic (arginine) and aromatic amino acids residues. The protein-dye complex causes a shift in the absorption maximum of the dye from 465 nm (red brown) to 595 nm (blue) (Fig. 88). Coomassie Blue binds roughly stoichiometrically to proteins, so this staining method is well suited for densitometric determinations. The proteins are detected as blue bands on a clear background, after fixing the gel with TCA for obtaining maximum sensitivity.

λ_max = 465 nm

λ_max = 595 nm

Coomassie Brilliant Blue G-250

Figure 88. Scheme of protein staining by Coomassie® Brilliant Blue G-250. Coomassie dye changes from a brown red cationic form to a blue anionic blue in the presence of protein.

Stock solution was prepared from: acetic acid 10 % (v/v); methanol 40 % (v/v); Coomassie Brilliant Blue G-250 0.1 % (w/v). First the gel was fixed for 30 minutes in fixing solution 12

% trichloroacetic acid (TCA) in MilliQ. Then the gel was shaken overnight with a mixture of 80 ml buffer (10 % (NH₄)₂SO₄, 2 % H₃PO₄ in MilliQ) with 20 ml methanol and 2 ml Coomassie Brilliant Blue G-250-Colloidal Concentrate, and afterwards the gel was washed with 25 % methanol for 60 seconds and scanned with a GS-710 Calibrated Imaging Densitometer from BIO-RAD.

3.6.4.3 Proteins extraction by passive elution

The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is mixed directly with the matrix solution and measured by MALDI-MS. Compared to other methods based on electroblotting or electroelution, this method is much simpler and less time consuming. The sensitivity of proteins detection by MS is higher than or comparable to the Coomassie Blue staining procedure for proteins up to

about 25 kDa. The use of solvents containing formic acid has been described for the elution of hydrophobic and hydrophilic peptides and proteins from SDS-polyacrylamide gels, yielding elution recoveries of 77 - 95 %.

I. 1-D SDS-PAGE was performed on precast Tris-tricine gels. After staining with Coomassie Blue, the gel was washed and destained in acetic acid/ethanol/H2O (1:3:6, v/v/v).

II. The protein spots were cut from the gel and dried in a Speed Vac evaporator. The gel piece was then incubated in 20 - 50 µl of an organic solvent mixture composed of formic acid/

acetonitrile/isopropanol/H₂O and macerated with a plastic pipette.

III. The incubation was performed at 35 °C in an ultrasonic bath for 30 min. A small fraction was diluted to a protein concentration of approx. 1 pmol/µl (assuming protein recovery of ca. 50 %) for MS analysis.

Figure 89. Schematic protocol of the treatment of passive elution.

3.6.4.4 Proteomic imaging system for unstained gel

The bioanalyzer gel reader (LaVision BioTec GmbH, Bielefeld, Germany) offers new perspectives in proteomic imaging system, as no dyes are required to visualize the protein spots ^[324]. The bioanalyzer gel reader utilizes native fluorescence of amino acids (mainly tryptophane) to visualize the proteins within the gel ^[325]. The outstanding advantage is time reduction as soon as the gel run is finished it can be imaged. Therefore the proteins can be conducted directly to downstream applications like blotting or MALDI-MS.

1-DE spots excision

Crash the gel

Sonication in ultrasonicbath – 10 to 30 min

Removal of Supernatant by centrifugation

MS anaysis

•Formic acid/acetonitrile/isopropanol/water (50:25:15:10 v/v/v/v )

The system utilizes UV extended white light illumination by a 300 W xenon lamp (265-680 nm), advanced filter techniques and parallelized photo multiplier detector. The high image acquisition speed of 1 cm²/s facilitates rapid scanning with a spatial resolution of 40 µm over the entire sample area. Depicting unstained protein amounts down to 1 ng has a superb sensitivity. After protein spots was fixed in position on the gel tray, localisation and isolation of gel spots was carried out by moving the gel tray, with positioning and scanning of the gel controlled by the LaVision-Biotec scanning software (Fig. 90).

Mirror

Imaging lens

Gel

XY Stage

Light guide

Xe-lamp

Photomultiplier

Figure 90. Scheme of the gel bioanalyzer adapted from http://www.lavisionbiotec.com/en/microscopy-products/gelreader/