SDS-PAGE employs a multi-phasic buffer system with differing pH to cause separation according to molecular weight. The samples are loaded into very narrow starting zones, which along with the discontinuities, determines the subsequent sharpness of the separations and is responsible for the very high resolving power of the multi-phasic buffer system. The advantage of this system is that large volumes of dilute protein samples can be applied to the gel without loss of resolution of their component
polypeptides. The pH of the Tris.HCl buffer used in the stacking gel has a different pH to the Tris.HCl buffer used in the resolving gel, which have been specifically selected
so that the proteins migrate at a pace between that of the leading Cl- ions and of the trailing glycinate ions. When the current is applied, a boundary is formed where the proteins collect in the stacking gel and then move according to molecular mass in the resolving gel. Lots of negatively charged SDS molecules bind to the hydrophobic regions of proteins, disrupting non-covalent bonds and cause unfolding. The intrinsic charge of the protein is masked, causing them to migrate to the cathode when the voltage is applied. Sample preparation involved measuring the protein concentration of each sample using Bio-Rad, to quantify against a standard curve, so as to set up samples with 25 µg of protein. If too much protein is loaded onto a gel, it can smear and
individual bands are hard to define. For volumes of substances to make up the resolving and stacking gels, refer to tables in Appendix. Gels were run at 180 Volts for 50
minutes.
2.6.2 Western Blotting
Western blotting procedures have been optimised to reduce background and decrease experiment time, using the method outlined here.
SDS-PAGE, Transfer and Overnight Block
Proteins were resolved by SDS-PAGE (10% acrylamide for HMW proteins) or Native PAGE against either High Molecular Weight Calibration Standards (Amersham) or His- tagged protein markers (Benchmark or MagicMark XP, Invitrogen) and the resulting gel was washed in transfer buffer (Transfer Buffer (1L) 5.8 g Tris base, 2.9g glycine, 200 mL MeOH, 0.05% SDS (500 mgs) (for membrane proteins and those > 80 kDa). Polypeptides were transferred from the gel to a PVDF membrane (Hybond-P, GE Healthcare) at a constant voltage of 120V in transfer buffer for 90 minutes, using a BioRad electroblotting kit. Before transfer, the membrane was activated by placing in methanol for 5-10mins, washing with deionised water then adding to transfer buffer for 2mins. After transfer, membranes were blocked in 50mL of 10% Milk powder in TBS containing 0.1% Tween-20 (TBST-T) and rocked for 1 hour at room temperature before being left stationary (but rocked if possible) in the cold room O/N.
Antibody Incubations
Wash the milk away with 50mL of TBS-T for 15min. The TBS-T wash step was repeated twice more. Primary Incubation: 20mL of TBS-Tween containing 5% milk, add 20µL of anti-his IgG mouse monoclonal antibody (Roche - binds to either His terminus) or 8µl anti-His IgG mouse monoclonal (N-terminal preferential antibody – Sigma H1029), and leave to shake for one hour. Follow with three 20mL TBS-T wash steps of 15 mins each. Secondary Incubation: 20mL of TBS-T containing 5% milk, add 10µl of anti-mouse IgG conjugated to horseradish peroxidase derived from sheep (Sigma, A5906) was added and left to shake for two hours before three further TBS-T wash steps. For the final wash step, membranes were left to rock overnight at RT to reduce the background of unbound proteins.
Development
For developing – in the dark room, 5mL of western blotting reagent 1 (GE Healthcare) and 5mL of western blotting reagent 2 (GE Healthcare), or more recently – 2 mL of EZ- ECL Solution A and 2 mL Solution B were added to the membrane and allowed to develop for 2 minutes (preferably in the dark). The developed membrane was held by tweezers to drip excess developing solution onto a tissue, and placed is a cassette lined with cling-film. Exposure time to x-ray films (Super-RX NIF, Fujifilm) varied from 30 seconds and 5 minutes, but sometimes up to 30 minutes. Observed bands resulted from electrochemical luminescence (ECL) from proteins embedded in the membrane. Latterly, instead of developing using Fuji Film, a chemi-illuminescence detector was used to analyse the Western Blot PVDF Membrane.
2.6.3 Protein Concentration Determination 2.6.3.1 BioRad Assay
The BioRad assay was used to measure the protein concentration of soluble protein samples of unknown concentration and was carried out as described by the BioRad protocol. The concentrated BioRad reagent was diluted 1 in 5 with ddH2O and 1 mL aliquoted in to plastic cuvettes. A standard curve was performed on each occasion,
using the sample volume of the protein sample of unknown concentration added as Bovine Serine Albumin (BSA), typically 1.5 µL. Each cuvette was inverted 3 times using parafilm as a cuvette ‘lid’, before measuring the absorbance at 595 nm. Protein samples were done in triplicate.
2.6.3.2 BCA Assay
A BCA (bicinchoninic acid) assay was used to measure the protein concentration of membrane protein samples of unknown concentration and was carried out as described by the Pierce protocol. BSA protein standards were made in 0.25 mg/mL increments from 0 to 2 mg/mL BSA. Reagents A and B were mixed at a 50:1 ratio, a volume sufficient for 9 x BSA standards plus the number of samples to test in triplicate). 1 mL of mixed BCA reagents was aliquoted into plastic cuvettes. 2 µL of each BSA standard was added to a cuvette, along with 2 µL protein sample of unknown concentration, always done in triplicate. Addition of protein samples was done accurately but with haste, as the colour change of the BCA reagents once mixed together, is time (and temperature) dependent. Parafilm was secured over the top off a cuvette and the cuvette was inverted three times to mix the protein sample with the reagent. Reactions were left to incubate at 37 °C in a static incubator for exactly 30 minutes. Absorbancies were measured against a blank (1 mL BCA A+B, + 2 µL water, i.e. 0 mg/mL BSA) at 562 nm using a using a Jenway 6306 UV-visible spectrophotometer. Protein concentrations were calculated based upon the standard curve. The working range for the assay is between 20 and 2000 µg/mL.