The purity of a potential scFv standard was analysed by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). This analytical method separates protein by molecular weight. Sodium dodecyl sulphate (SDS) is a surfactant that denatures the proteins and remains bound, producing an overwhelmingly negative charge. Unlike native electrophoresis, separation by SDS-PAGE is entirely due to molecular weight and not the protein’s charge or its protein structure.
In order to achieve efficient separation of the scFv fragment, a discontinuous gel was used, in which there was a stacking gel placed on top of the separating gel. The stack
Samples were prepared by diluting them 1:1 in sample buffer (60 mM Tris-HCl pH 6.8, 10% Glycerol, 2% SDS (GibcoBRL, Life Technologies Inc., Paisley, UK), 0.001% Bromophenol Blue (Sigma-Aldrich Company Ltd.) and 5% P-mercaptoethanol). Then samples were denatured by boiling for 3 minutes. SDS-PAGE was performed using a dual mini-slab gel system (Atto System AE 6400, supplied by Genetic Research Instruments Ltd., Essex, UK). The glass plates were cleaned using Industrial Methylated Spirits (IMS), and gloves were worn to prevent contamination with external proteins. Once cleaned the plates were set up as in the manual.
The separating gel was prepared as in Table 6.1. Gloves had to be worn at all times as most of the reagents are toxic. Separating gel was degassed to remove any air bubbles, by passing it through a 0.2 pm polysulfone syringe filter (Whatman Ltd.). Polymerisation o f the gel was initiated with the addition of 225 pL of freshly made 10% ammonium persulphate and 15 pL of NNN^N’-Tetramethylethylenediamine (TEMED). The gel was carefully mixed whilst avoiding bubbles, then poured into the space between the two plates and allowed to set. Water was poured on to the setting gel to prevent it from drying out.
Table 6.1 SDS-PAGE separating and stacking gel formulations (adapted from Hames, 1990)
Reagent Separating Gel (mL) Stacking Gel (mL)
Acrylamide-bisacrylamide (30:0.8) 6.25 1.25
Tris-HCl 3.0M (pH8.8) 1.875 -
Tris-HCl 0.5M (pH6.8) - 2.5
10% SDS 0.15 0.1
Distilled Water 6.125 5.0
Above values will prepare x2 mini gels. Protogel (National diagnostics, East Riding, UK) a ready-made liquid form o f Acrylamide-bisacrylamide was used.
Once the separating gel had set, the water was poured off. The stack was prepared as in Table 6.1 and degassed as before. Polymerisation was initiated by the addition of 150 pL o f 10% ammonium persulphate and 15 pL of TEMED. Again the gel was carefully mixed avoiding bubbles, then poured on top of the separating gel. A comb was carefully inserted between the two plates, and the gel was then left to set. Once the gel had set, the comb was carefully removed, so as not to destroy the formed wells. The glass plates were placed in the electrophoresis tank. To the tank 1/10 dilution of reservoir buffer (0.25M Tris, 1.92M glycine, 1% SDS, pH 8.3) was added. Wells were flushed with reservoir buffer to remove any residue gel.
Appropriate volumes (10 pL) o f the denatured samples (boiled for 3 min) were loaded in to the wells. To the first two wells, a low molecular weight marker (Amersham Pharmacia Biotech) and scFv standard were added respectively. The tank was connected to a power supply. Separation through the stack was at 64 V and 30 mV (30 min). Once the samples had reached the separating gel, signalled by the formation of a single blue line across the gel, the power was increased to 120 V and 50 mV. The separation was run for a further hour, until the blue line reached the bottom o f the plate.
Gels were either stained using Coomassie brilliant blue (Sigma-Aldrich Company Ltd.) or Western blotted. Gels were stained by gently agitating on a rocking table (Luckham 4RT rocking table, Denley Instruments, Billinghurst, UK) for 1 hour in staining solution (Coomassie brilliant blue R-250 (1 gL'^) (Sigma-Aldrich Company Ltd.) in 500 mL water, 400 mL methanol, 100 mL acetic acid). Then the gels were transferred into destain solution (25% methanol, 10% acetic acid in distilled water) and continued agitation until protein bands were revealed.
If the samples were to be Western blotted, the protein had to be transferred from the gel on to Immobilon-P PVDF transfer membrane (Millipore (UK.) Ltd., Watford, UK). Transfer was performed by a Trans-Blot semi-dry transfer cell (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The gel was carefully removed from the glass plates and placed in 50 mL of Blotting buffer (water 700 mL, glycine 2.92 g. Tris 5.8 g, SDS 0.374 g, methanol 200 mL) for 10 min. Gloves were worn when manipulating the gel. The anode plate of the Trans-Blot semi-dry transfer cell was wetted with blotting buffer. Two sheets of filter paper moistened with blotting buffer were laid on top of the anode. The Immobilon-P PVDF membrane was cut to the desired shape, then moistened with methanol followed by blotting buffer. This was placed on top of the filter paper. A sterile pipette was carefully rolled over the membrane to remove air bubbles. On top of the Immobilon-P PVDF membrane, the gel was carefully laid, avoiding air bubbles. One more piece of moist filter paper was placed on top o f the gel. The cathode was moistened with blotting buffer and placed on top o f the unit. Transfer took 30 min operating at 20 V.
After the samples were transferred, the immunoassay was commenced. To ensure the reaction occurred evenly over the whole of the membrane, the vessel was gently agitated on rocking table (Denley Instruments) throughout the protocol. The membrane was blocked using 0.5% milk powder in PBS for 1 h. Casein in the block prevents non specific binding to the membrane. The membrane was then incubated overnight with rabbit polyclonal antibody directed against the anti-HEL Fv fragment, at 1 in 2000 dilution in PBS with 0.1% milk powder. After incubation the membrane was washed for 10 min in distilled water, followed by 10 min in PBS with 0.1% milk powder, this was repeated twice. After washing, the membrane was incubated for 2 h, with blotting grade Goat anti-rabbit IgG conjugated with Horse Radish Peroxidase (Bio-Rad) at 1 in 2000 dilution in PBS with 0.1% milk powder. The membrane was washed as previously described. Colour was developed by incubating the membrane in a solution o f 34 mL PBS, 6 mL methanol, 20 mg 1 -chloro-4-naphthol (Sigma-Aldrich Company Ltd.) and 20 pL o f 30% H2O2 (Sigma-Aldrich Company Ltd.) for 15 min. Once bands had