stacking gel (3ml) was poured to give better resolution of larger sample volumes. Various slot formers were inserted to give either 10/15 slots, or a preparative comb with marker slot.
Samples for electrophoresis were diluted with one fifth volume of 5x Laemmli
sample buffer (Bio-Rad; 62.5mM Tris.HCl(pH6.8) / 2%(w/v) S D S / 10%(v/v)
glycerol/50mM /3-mercaptoethanol/0.001%(w/v) bromophenol blue) and denatured at 95°C for 4 mins. Various volumes up to a maximum of 40/il were loaded per slot after centrifugation for 30s at RT. Electrophoresis was performed at 180V for 40-50mins in Ix gel running buffer (25mM Tris/ 192mM glycine/ 0.1%(w/v) SDS).
Molecular weight markers were electrophoresed in adjacent lanes. These were
either standard protein markers (Sigma, 6SDS) or pre-stained markers when gels
were to be Western blotted (Appendix E).
2.4.13: Coomassie blue staining
After electrophoresis the gel apparatus was dismantled and the right-hand comer of gel was cut-off with a razor blade. The gel was then transferred to a small square dish of Coomassie Brillant Blue R-250 (0.1%, w /v) in destain (40%(v/v) methanol/10% (v/v) glacial acetic acid in ddHjO) and agitated for 1-2 hrs at RT. The stain was then removed and the gel was successively rinsed with destain alone until a clear background was obtained.
Stock Coomassie Brillant Elue R-250 (0.1%; w /v) was prepared in the fumehood by dissolving the powdered dye in destain and then filtering the solution through 3MM filter paper.
2.4.14: Protein transfer to nitrocellulose - Western blotting (Sartorius or Bio-Rad, Semi-dry blotters)
Western blotting was carried out according to the method of Towbin et a l
(1979) using either the Sartoblot-IIS (Sartorius) or Trans-blot SD (Bio-Rad), semi-dry blotters.
After SDS-PAGE gels were transferred to Ix blotting buffer (48mM Tris/ 39mM glycine/0.38%(w/v) SDS/20% (v/v) methanol) equilibrated to 4°C for 15- 20 mins. Five or six sheets of 3MM paper were then wetted in the same buffer and placed on the base plate of the blotter (graphite cathode of Sartoblot-IIS, or, platinum anode of Trans-blot SD). A sheet of nitrocellulose of the appropriate size was then wetted in the buffer.
For the Sartoblot-IIS, the gels were then placed on top of the 3MM paper and the soaked nitrocellulose placed on top taking care to remove all air bubbles. In the case of the Trans-blot SD, the nitrocellulose was placed on the wet 3MM paper and then the gels positioned on top of the wet membrane. In both cases the sandwich was completed by placing a further five or six sheets of wetted 3MM on the apparatus and replacing the lid (graphite anode of the Sartoblot-IIS, or. Stainless steel cathode of the Trans-blot SD). Blotting was performed at RT for 1 hr. In the case of the Sartoblot-IIS, this was at 100mA constant current whereas for the Trans-blot SD this was at 15 V constant voltage. After transfer, blots were allowed to dry overnight at RT between 3MM paper.
Prestained molecular weight markers were transferred along with the other proteins and their positions marked with a blue ballpoint pen. These were later used for estimating the size of immunoreactive bands.
2.4.15: Immunostaining of Western blots
Non-specific binding sites were blocked with PBS (Phosphate buffered saline, Dulbecco ’A ’; lOmM sodium phosphate(pH7.3)/150mM NaCl/4mM KCl, tablets
from Oxoid)/5% non-fat milk (’Marvel’) or PBS/3% (w/v) BSA for 1 hr at RT or over-night at 4°C. Blots were then rinsed with PBS/0.1% (v/v) Tween 20 (polyoxyethylenesorbitan monolaurate; PBS-T), and then incubated with rabbit antisera or affinity purified /z-Chimaerin antibodies diluted in PB S/l% (w /v) Marvel (or BSA), for 1-2 hrs at RT or overnight at 4°C. The filter was then washed at RT with PBS-T (6x, 5-10 mins) and then incubated for 1 hr at RT with anti-Rabbit immunoglobulins conjugated with Horseradish peroxidase (HRP) diluted 1:500 in PB S-T /l% (w /v) Marvel. The colour was developed using PB S/ 0.025%(w/v) diaminobenzidine tetrahydrochloride (DAB, Sigm a)/0.03%(w/v)
hydrogen peroxide (H2O2, BDH).
2.4.16: Enhanced ChemiLuminescence (Amersham, Research News, 3(5), 1989)
In September 1989, Amersham started marketing their Enhanced
ChemiLuminescence system for the non-radioactive labelling and detection of DNA. This involved the cross-linking of a modified horseradish peroxidase (HRP) to denatured D N A and following hybridization, the incubation of membranes in detection reagent. The peroxidase complex bound to the probe stimulates the production of a peroxide ion from the peracid salt in the detection solution. The peroxide ion then reacts with luminol and an enhancer, which results in a sustained emission of blue-light for up to 60 mins.
The similarity between the method described and that of conventional antibody- immunoglobulin:immunoglobulin-HRP complexes being used to form coloured products, led me to enquire about the availability of the reagents for use in Western blotting.
The methods for immunostaining were followed exactly as for detection with DAB, except that primary antibodies were used at 10-20 fold higher dilutions, secondary antibodies conjugated with HRP were diluted 1:1000-1:2000 and after the final wash in PBS-T, nitrocellulose filters were rinsed in PBS before transferring to the detection reagents.
An equal volume of detection reagents 1 and 2 were mixed in a 10ml sterilin tube or 50ml falcon tube (approximately 5ml per filer). This was then poured into
Hyperfilm-ECL Enhancer A m in o p h th a h c A cid Peracid salt 2(Hpj 2H,0 Secondary Ab P rim ary A b Antigen # Luminol
N itro cellu lo se Filter
a flat-bottomed dish (Sterilin). Nitrocellulose filters were then immersed successively for 1 min each in the combined reagents, wrapped in saran wrap (Dow) and then exposed to film (Hyperfilm-ECL; Amersham) at RT for 40s, 2-5 mins or up to 60 mins.
2.4.17: Antibody production in Rabbits: Polyclonal sera (Harlow and Lane, 1988)
New Zealand White rabbits (2-2.5kg) were injected with lOO^g of protein per immunization. For the first injection protein dissolved in PBS was added to an equal volume of Freund’s Complete Adjuvant. This was well vortexed until a complete emulsion was formed. Animals were then injected in a single spot subcutaneously. All subsequent booster injections were given using Freund’s Incomplete Adjuvant at 4-6 week intervals over a period of almost two years. Rabbits were bled 10 days after each booster immunization by cutting the ear vein with a razor blade.
20-40ml of blood was collected in a 50ml Falcon tube and allowed to clot at RT or overnight at 4°C. The clot was then broken up using a pasteur pipette sealed to form a glass rod. The serum was then obtained by centrifugation at 4°C, 3,000rpm for lOmins (MSE, Coolspin2) and then decanting the supernatant to a fresh tube. Sodium azide was added to serum at 0.1%(w/v) final concentration and then aliquots stored at -20°C.
2.4.18: Coupling of Bacterial Proteins to CNBr-activated Sepharose 4B
(Pharmacia; Hoffman et a l, 1987)
Preparation of sepharose: l-3g of CNBr-activated Sepharose 4B powder (Sigma) was swelled with ImM HCl for 10 mins at RT. The matrix was washed on sintered glass with a further 250ml of ImM HCl, filtered until dry, and then transferred to a 50ml falcon tube.
Preparation of protein: jS-Gal fusion proteins in 8M urea were dialysed
extensively against ddHjO. Insoluble fractions of trpE or trpE fusion proteins were
precipitated with 5 volumes of acetone over-night at -20°C, in Corex tubes. Acetone precipitated proteins were spun down at 4°C, 15,000rpm for 15 mins
(Beckman J2-21) then dried and dissolved in coupling buffer (O.IM NaHCO^/ 0.5M NaCl, pH8-9) or PBS. 5-lOmg of proteins dissolved in PBS or dialysed against ddHjO ()3-Gal fusions) were then dissolved with an equal volume of 2x coupling buffer and the volume then increased with Ix coupling buffer. When necessary 0.1%(w/v) SDS (final conc.) was included to aid the complete dissolution of the protein.
Protein dissolved in coupling buffer was transferred to the 50ml falcon tube, containing the previously prepared sepharose gel. The gel-ligand suspension, in a final volume of 40ml, was mixed end-over-end overnight at 4°C.
The following day the gel was applied to a sintered glass and the solution removed by filtration. This was retained until it was ascertained whether or not the protein had in fact bound to the matrix. This was then washed three times alternately with O.IM NaOAc(pH5,2)/0.5M NaCl followed by coupling buffer. Unreacted groups on the sepharose were then blocked with 0.2M glycine in coupling buffer, by transferring the washed matrix to a fresh 50ml Falcon containing 40ml of buffer. This was mixed by rotating end-over-end for 3-4 hrs at RT, or on rare occasions over-night at 4°C. The matrix was finally made ready by applying it to an OMNIFIT column and washing it extensively with coupling buffer followed by PBS.
2.4.19: Affinity purification of Antibodies from sera
Purification of n-Chimaerin-specific Antibodies on matrix bound protein (Carroll and Laughon, 1987; Segev et a l, 1988)
An omnifit column loaded with Sepharose 4B-bacterial protein was equilibrated using 50ml o f PBS, at a flow rate of 0.5ml/min. Rabbit serum to be purified was diluted 1:1 with PBS and loaded onto the column. This was then recycled for 1-2 hrs at RT.
The flow through (FT) was collected and stored on ice. The column was then washed successively with 50ml of PBS, 50ml of BBS-T (O.IM boric acid/ 25mM sodium borate/IM NaCl/0.1% (v/v) TWEEN 20) and 50ml of PBS. The bound antibody was then eluted with 0.5M acetic acid and neutralized by the addition
of 0.05 volume of 0.5M phosphate buffer. Bovine albumin (Sigma, >98% GE) was added to 0.1%(w/v) and the antibodies then dialysed against 3 litres of PBS at 4°C overnight.
The column was regenerated by washing with 50ml of O.IM sodium acetate (pH5.2)/0.5M NaCl followed by re-equilibration with PBS/0.1% sodium azide.
Throughout the antibody purification ODjgo readings were taken o f all of the fractions coming off the column.
2.4.20: Purîfîcation of Immunoglobulins from whole serum (Harlow and Lane, 1988)
Rabbit serum was centrifuged at 4°C, 3,000rpm for 30 mins (MSE, Coolspin2). The supernatant was transferred to a beaker and stirred with a magnetic stirrer. While stirring gently, one volume of saturated ammonium sulphate (761g in 1000ml, pH7.0) was added slowly at RT and then stirring continued overnight at 4°C. The precipitate was pelleted by centrifugation at 4°C, 3,000rpm for 30 mins (MSE, Coolspin2). The supernatant was discarded and the pellet drained well. The pellet was resuspended in 0.5 volume (of the starting volume) of PBS. This was then dialysed against two changes of 2 litres of PBS overnight at 4°C. The antibody preparation was then used for the affinity purification of «-Chimaerin- specific antibodies.
2.4.21: Antibody production in Mice: sera
BA LB/c mice (3-4 months old) were injected subcutaneously with 50/ig of protein per immunization, using the same regime as that for rabbits. For the first injection protein dissolved in PBS was added to an equal volume of Freund’s Complete Adjuvant. All subsequent booster injections were given using Freund’s Incomplete Adjuvant at 4-6 week intervals over a period of 4-6 months. Test bleeds were performed 10 days after each booster immunization by cutting a piece off the warmed tail using a razor blade. The final boost was administered intraperitoneally using PBS and no adjuvant 4-7 days before ’fusion’.
Approximately 0.5ml of blood was collected on the day of ’fusion’ when animals