Electrophoresis and concentration determination Laemmli SDS-PAGE gel electrophoresis Laemmli SDS-PAGE gel electrophoresis

After each purification step, samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970) which separates proteins according to their molecular weight. For an expression analysis, 200 µL samples of the uninduced and harvested cultures were spun down at 13000 rpm for 1 minute at RT.

The supernatant was removed and the pellet was resuspended in 80 μL water to which 40 µL of 3x loading buffer (10 ml contained 0.9 g SDS, 3 g glycerol, 1.8 mL 1M Tris pH 6.8, 1mL β-mercaptoethanol) was added. The β−mercaptoethanol reduced disulfide-bridges.

During protein purification, 80 µL samples of the cleared lysate (supernatant), pellet resuspended in water, and flow-through during supernatant injection onto the column were collected and combined with 40 µL water and 60 µL 3x loading buffer.

Similarly, 20 µL samples of the elution fractions were combined with 10 µL 3x loading buffer. Samples were then heated for 3-5 min at 95 °C.

The SDS-PAGE gel consists of two parts: a 3.75 % stacking gel and 5 – 15%

separating gels. 12% and 15% separating gels were used in this study (Table 2.6). For the combs to make 15 wells/gel, 10 µL protein samples were loaded into each well of the stacking gel. The gels were run with at 150-200 V using the Mini-Protean 3 Electrophoresis Cell system from Biorad using Laemmli running buffer (250 mM Tris, 1.9 M glycine, 1% SDS). The SDS coating on the proteins gave it a charge:mass ratio causing the protein to travel down the gel toward the positive electrode when an electric field is applied. The proteins moved quickly through the stacking gel; however, they were slowed when they reached the edge to the separating gel. Due to the pH difference between the

stacking and separating gels, the sample became concentrated at the border, which sharpened the bands. Heavier proteins move more slowly through the pores than smaller proteins. Increasing the concentration of polyacrylamide in the separating gel resulted in better resolution of large proteins.

Table 2.6: Laemmli SDS-PAGE gel components

Stacking gel 3.75% Separating gel 12% 15%

Upper gel buffer (pH 6.8) 2.5 mL Lower gel buffer (pH 8.8) 2.5 mL 2.5 mL

dH20 6.14 mL dH20 3.4 mL 2.4 mL

30% AMBA 1.25 mL 30% AMBA 4.0 mL 5.0 mL

TEMED 10 μL TEMED 5 μL 5 μL

10% APS 100 μL 10% APS 100 μL 100 μL

*makes 4 gels *makes 2 gels

Upper gel buffer (0.5 M Tris, 0.4% SDS)

Lower gel buffer (1.5 M Tris, 0.4% SDS

AMBA=acrylamide/N,N'-methylene-bis-acrylamide (30% acrylamide, 0.8 % bisacrylamide stock) APS= ammonium persulfate (10% stock)

TEMED=tetramethylethylenediamine

Schägger Jagow SDS-PAGE gel electrophoresis

Schägger Jagow gel electrophoresis (Schägger 1987) can separate proteins in a total range from 1 to 100 kDa with the optimal resolution for proteins smaller than 30 kDa.

The use of tricine in the cathode buffer has a lower negative charge than glycine used in the Laemmli SDS-PAGE running buffer. Its higher ionic strength causes it to travel faster through the gel. This results in a higher resolution of proteins ≤10 kDa because the ions move more quickly through the lower part of the gel than the low molecular weight proteins, even at lower polyacrylamide concentrations. This system consists of both a separating gel on the bottom and a stacking gel on the top (see Table 2.7). Protein samples to be loaded onto gels were combined with 2x Schägger Jagow sample buffer (50 mM Tris to pH 6.8 with HCl, 4% SDS, 0.01% Serva Blue G, 12% glycerol, 2% β-mercaptoethanol) and boiled for 3-5 min at 95°C. Cathode buffer (0.1 M Tris, 0.1 M Tricine, 1% SDS) was placed on the inner portion of the apparatus between the glass plates and anode buffer (0.2 M Tris, pH 8.9) was placed on the outside of the glass plates. The Mini-Protean 3 Electrophoresis Cell system from Biorad was utilized. The gels were run with at 70 V until the proteins were in the separating gel then they were run at 120 V for the remainder.

Table 2.7. Schägger Jagow gel components

4% Stacking gel 10% Separating gel

30% AMBA 400 μL 3.32 mL

Gel buffer 750 μL 3.35 mL

ddH2O 1.85 mL 1.14 mL

50% glycerol --- 2.12 mL

TEMED 4 μL 6 μL

10% APS 20 μL 50 μL

*makes 2 gels

Gel buffer=3M Tris adjusted to pH 8.45 with HCl, 0.3% SDS AMBA=Acrylamide/N,N'-methylene-bis-Acrylamide

AMBA=30% acrylamide stock solution with 0.8 % bisacrylamide APS=Ammonium persulfate

TEMED=Tetramethylethylenediamine

Molecular markers and staining/destaining solutions

A 15% SDS PAGE is suitable to analyze proteins with molecular weights between 10 to 100 kDa. Molecular weights of the bands were estimated using various molecular markers from Fermentas (catalog no. SM0431, SM0671, SM1861, SM1881) and Sigma (M3913). Gels were stained with Coomassie Brilliant Blue solution (0.25% w/v Coomassie Blue, 7.5% acetic acid, 40% ethanol) by boiling it for one minute in a microwave. After cooling to RT , the gel was moved into a destaining solution (10%

acetic acid, 40% ethanol) and left shaking overnight. A faster variation of this adopted from the Prof. Brose lab (Goettingen, Germany) involves staining solutions A (500 mg CoomassieR, 650 mL H20, 250 mL isopropanol, 100 mL acetic acid), B (50 mg CoomassieR, 800 mL H20, 100 mL isopropanol, 100 mL acetic acid), C (20 mg CoomassieR, 900 mL H20, 100 mL acetic acid), and destaining solution D (900 mL H20, 100 mL acetic acid). The gel was covered with staining solution A and heated for 1 min in a microwave. Staining solution away was removed and staining solution B was used to cover the gel which was then heated in the microwave for 1 min. This procedure was repeated for staining solution C followed by destaining solution D. The gel was then left shaking gently at RT for 15-30 min before the destaining solution was removed and replaced with water. Visualization within 40 min was sometimes achieved using PageBlue Protein Staining Solution (#R0571) from Fermentas.

Measurement of protein concentration

Protein concentrations were usually determined spectroscopically using an ND-1000 spectrophotometer from NanoDrop Technologies Inc., Rockland, USA which used a path length of 0.2 mm and measured the absorbance of aromatic amino acid residues at a

wavelength of 280 nm using the molecular weight and extinction coefficient of the protein.

For proteins like Rab33BQ92L which had GTP bound which absorbs at 260 nm, protein concentrations were determined using the Bradford assay (Bradford 1976) to avoid interference of nucleotide absorbance. A standard curve was calculated based on 1, 2, 3, 4, 5, 7, 8, 9, and 10 µg BSA. This protein was added to sterile water with a final volume of 200 µL. 800 µL of Bradford working solution (500 mL contained: 425 mL dH20, 15 mL 95% ethanol, 30 mL 88% phosphoric acid, 30 mL Bradford stock {100 mL 95%

ethanol, 200 mL 88% phosphoric acid, 350 mg Serva Blue G) which is in its protonated form and has a reddish-brown in color was added to the 200 µL standard protein. Protein binding stabilizes the dye in its anionic state resulting in a color change to blue, and the intensity depends on the concentration of protein. The absorbance of the mixture was measured at 595 nm on a Genesys 6 spectrophotomer (Thermo Spectronic). The concentration of the protein was determined from the BSA standard curve.