CHAPTER 1 INTRODUCTION
2.2 Immunochemical techniques
2.2.1 Western blotting
functions, protein functional classifications are based mostly on domains rather than complete proteins.Pc96 and other similar proteins were used to screen the PROSITE database. Certain functional domains contain sets of conserved regions of amino .acids, and the occurrence and positions ofthese regions along the length of a protein sequence, in relation to certain alignment features can be used to create a signature of the domain, allowing the identification of these patterns within query sequences.
These signatures are a reflection of the 3-dimensional conformation of the domain, and can be used to assign function to regions of un-characterised proteins, providing the profile of that region is a significant match.
The 3-dimensional structure and function of protein sequences was also determined by analysis with the 3D-PSSM (Three Dimensional - Position-Specific Scoring Matrix) server. The server contains a database with known protein structures, which are compared to the query sequence and scored on a basis of compatibility. Factors such as secondary structure elements, and probability of occupying various states of hydrophilicity, in relation to overall shape are analysed (Kelleyet al., 2000).Entries in the PROSITE database make up the so-called BLOCKS database, used to identify families of proteins. This is not a comparison of the sequence itself, due to the fact that homologous proteins may not share sequence similarity. A block relates to motifs, or conserved stretches of amino acids conferring specific function to the structure of a protein. Individual proteins may contain several blocks in similar combination, corresponding to specific structure or function. The query sequence is aligned against all blocks in the database at all available positions. A score is derived from alignments using the position-specific scoring matrix (PSSM), taking into account matches at given positions and the probabilities of amino acids occupying specific positions in the block (Baxevanis, 1998).
electrophoresis (SDS-PAGE) and transferred electrophoretically to nitrocellulose.
Nitrocellulose is a matrix with a high affinity for protein. The method used in this study was that of Towbin et al.(1979), with minor modifications. The transfer buffer included methanol to enhance the binding of protein-SDS complexes to the membrane (Goodenham, 1984). The unoccupied sites on the membrane were blocked with non- fat milk. Antigens transferred onto the nitrocellulose binds to primary antibody, which in turn binds to secondary antibody specific for the primary antibody.
The secondary antibody contains a detection system with labelled enzyme horseraddish peroxidase (HRPO). HRPO catalysed a reaction involving a chromogenic substrate, forming a visual, insoluble precipitate. These effectively labelled specific bands bound to the nitrocellulose.
2.2.1.1 Materials
10% Cm/v) SDS. SDS (10 g) was dissolved in 100m1dist. H20 with gentle heating if necessary.
Blotting buffer. Tris (27.23 g) and glycine (64.8 g) were dissolved in 3.5 litres of dist.Hjf) and methanol (900 ml) was added. The volume was made up to 4.5 litresina large beaker. Prior to use, 10% (m/v) SDS was added (4.5 ml).
Tris buffered saline CTBS; 20 mM Tris, 200 mM NaCl, pH 7.4). Tris (2.42g) and NaCI (11.69 g) were dissolved in 950 ml of dist.Het), adjusted to pH 7.4 with He1, and made up to 1 litre.
1% Cm/v) Ponceau S. Ponceau S (0.1g) was dissolved in 1% (v/v) glacial acetic acid (100 ml).
Blocking solution [5% Cm/v) non-fat milk powder]. Low fat milk powder (5 g) was dissolved in TBS (100 ml).
0.5% Cm/v) BSA-TBS. Bovine serum albumin (0.1 g) was dissolved in TBS (20 ml).
4-chloro-I-naphthol substrate solution [0.06% Cm/v) 4-chloro-1 -naphthol, 0.0015%
(v/v) H2
ilil
4-chloro-1-napthol (0.03 g) was dissolved in methanol (10 ml). 2 ml of this solution was diluted to 10 ml with TBS, with the addition of 30% hydrogen peroxide (4 !-Ll).2.2.1.2 Method
After SDS-PAGE had been performed on duplicate gels, one gel was used for staining as a reference for the blot, and the other was used for blotting onto nitrocellulose. The nitrocellulose was cut to a size similar to that of the SDS-PAGE gel and floated on blotting buffer to soak. Six pieces of Whatman No. 4 filter paper were cut slightly larger than the nitrocellulose.A sandwich was constructed (Figure 2.1) with blotting buffer to avoid the entrapment of air bubblesthat can disturb the blotting pattern.
Two white Scotch-Brite pads, filter paper and the nitrocellulose and gel were positioned as seen in Figure 2.1. The sandwich was placed into a plastic support, into the western blotting apparatus and immersed in cold blotting buffer. The nitrocellulose was kept on the anodal side of the gel and blotting was achieved at 200 mA for 16 h. The blotting tank was placed in a larger container of ice. Blotting buffer . was stirred by a magnetic stirrer throughout the procedure to keep the distribution of cooling even.
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Direction ofprotein transfer
Figure 2.1 Arrangement of nitrocellulose sandwich prior to electro-transfer of protein. 1 - Scotch Brite pads; 2 - 3 x filter paper; 3 - SDS-PAGE gel; 4 _ nitrocellulose membrane.
The gel was removed and stained with Ponsceau S (l0 ml) in a glass petri dish to ensure that protein transfer had occurred. The nitrocellulose was removed and rinsed with dist H20 until the dye is removed.The nitrocellulose was air-dried for 1.5 h. The membrane was then blocked for 1 h with 5% (m/v) low fat milk powder in TBS. It was then washed in TBS (3 x 5 min), followed by a 2 h incubation with primary antibody in 0.5% BSA-TBS. A washing step was repeated (3 x 5 min) and followed by incubation in secondary antibody with HRPO enzyme conjugate in 0.5% BSA- TBS for 1 h. 4-chloro-l-naphthol substrate solution (lO ml) was used as a substrate for the development of the chromogenic reaction.
2.2.2 Screening for recombinants using DIG nucleic acid hybridisation and