Materials and Methods
3. Protein Analysis.
3.1 Protein Purification - total cell extracts.
Total cell protein extracts were generated using a high salt lysis buffer. A single cell suspension (10^-10^ cells) from tissues or from cell culture was first washed in ice cold PBSa. The cells were pelleted by centrifugation at 14k rpm for 10 seconds, and the supernatant was removed. The cell pellet was then resuspended in 300/d of protein extraction buffer I. The suspension was then frozen on dry-ice and thawed at room temperature three times, followed by a centrifugation at 14k rpm at 4**C for 10 min. The supernatant was taken and stored at -70**C until required.
3.2 Protein purification - nuclear/cvtoplasmîc extracts.
A single cell suspension (10^-10^ cells) was washed with ice cold PBSa. The cells were then pelleted (14k rpm 15 sec), and the supernatant removed to leave a combined cell/supematant volume of 100/il. The cells were then snap frozen in liquid nitrogen. On thawing, the cells were gently resuspended in 300/d of ice cold protein extraction buffer
n.
The suspension was centrifuged at 14k rpm for 15 seconds and the supernatant removed. This supernatant contains cytoplasmic proteins and can be stored at -70*C if required. The pelleted cell nuclei were then gently resuspended in 400/d of protein extraction buffer II, and centrifuged at 14k rpm for 15 seconds at 4"C, the supernatant being discarded. Again the pellet was resuspended in 200/tl of protein extraction buffer II. To this, lO/xl of 5M NaCl was added. After mixing well the sample was left on ice for 45 min. The sample was then centrifuged at 14k rpm at 4®C for 25 min. The supernatant, containing nuclear proteins, was removed and stored at -70"C until required.^ P 8 S a - Alt C
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3.3 Immunoprécipitation.
Single cell suspensions from thymus and spleen of transgenic and non-transgenic mice were prepared. Erythrocytes were removed by lysis in tris-buffered ammonium chloride (Sigma). Extracts were prepared by lysing cells in 1ml of immunoprécipitation buffer I on ice for 15 min. The extracts were centrifuged at 14k rpm for 5 min at 4®C and the supernatant was removed. To this, 50/tl of normal rabbit serum was added for 1 hr at 4®C, followed by 100/d of a
suspension of protein A-sepharose beads in lysis buffer for 30 min, and centrifugation at 14k rpm for 15 min at 4®C. Anti-Nef antibodies (HIV-1 HXB3 Nef antisera, to the N and C termini (Hammes et al., 1989) were then added at a 1:250 dilution to the cell lysate (supernatant), and incubated overnight on ice. Following this, the extracts were incubated again with lOOfil of 10% suspension of protein-A-sepharose beads for 1 hr. The beads were collected by centrifugation for 15 min. at 4”C and washed three times in lysis buffer. Pellets were resuspended in reducing sample buffer, heated at 100^ for 5 min. and the supernatants recovered. They were stored at - 70®C until required.
3.4 Protein Quantitation.
Using non-acetylated BSA (2mg/ml), standard protein concentrations of 0, 100,200,300, 500 and 1000/ig/ml were made in HgO. 5fi\ of the protein samples, or 20^1 of the protein standards, were added to 1ml of Coomassie protein assay reagent (Pierce), and mixed well. An HgO blank was then inserted into the spectrophotometer and the absorbance at 595nm was set to zero. The absorbance of the protein samples was then read at 595nm, with the absorbance of the 0/xg/ml standard being subtracted from each reading. Using a standard curve the concentration of the samples can then be calculated.
3.5 Western blot analysis of protein.
Gels were cast and run using the Bio-rad Western apparatus. The plates were cleaned, one was siliconised, the spacers were introduced and the plates clamped in place. The resolving gel was poured first. For 10ml of a 10% gel, this consists of 4ml HgO, 3.3ml of protogel acrylamide mix (30% acylamide and 0.8% bisacylamide), 2.5ml of 1.5M Tris pH8.8, 0.1ml of 10% SDS,
0.1ml of 10% ammonium persulphate, and 40/a1 of TEMED. After pouring, the gel was covered with 1:1 isopropanol/water (~ 0.5ml) to give a level surface to the top of the gel and to aid polymerisation. Once set the isopropanol/water is removed and the plates are dried. The stacking gel is then poured, and the comb is fitted. For 3ml of a 5% stacking gel, this consists of 2.1ml HgO, 0.5ml of protogel acrylamide mix (30% acylamide and 0.8% bisacylamide), 0.38ml of l.OM Tris pH 6.8, 0.03ml of 10% SDS, 0.03ml of 10% ammonium persulphate, and 30/d of
TEMED. The gel is not pre-run.
The protein samples and molecular weight markers are diluted 1:1 in 2x Western sample buffer, boiled for 5 min, centrifuged for 5 seconds and chilled on ice. 10-50/tg of protein is then run per lane. The gel is run at 5V/cm until the bottom dye reaches the bottom of the gel. After running the top plate is removed and the stacking gel is cut away. The gel is then floated off the bottom plate in Western transfer buffer, onto a piece of pre-wet Hybond C Extra (~ 10cm x 10cm). The transfer apparatus is then assembled. A pre-wet scotch-brite pad is covered with 2 sheets of 3MM paper, then the membrane and gel, 2 more sheets of 3MM paper and a second pre-wet scotch-brite pad. This sandwich is then placed in the transfer tank with approximately 3.5 litres of Western transfer buffer. The transfer of protein to the membrane for 3-5hr at 80V
320mA) is then performed at 4**C, with continuous circulation of the buffer.
3.6 Western blot hybridisation.
The membrane is first blocked for 2hr at 37®C with 50ml of Western block buffer. After 3x washes for 10 min with 50ml of PBS/0.1% Tween 20, the primary antibody is added. 20ml of block, with 2/ig/ml of the primary antibody is used, with an incubation at 37®C for 2hr. After 3x washes for 10 min with PBS/0.1% Tween 20, the secondary antibody (1:1000 - HRP conjugated), was added in 20ml of block solution. After 2hr at 37*C, 3x washes for 10 min with PBS/0.1% Tween 20 were performed, with the membrane stored under PBS/0.1% Tween 20 at 4®C until ECL™ detection.
3.7 ECL™ detection of Western blots.
The ECL™ detection system (Amersham) was used to visualise antibody binding to Western blots. In this protocol, 2ml of EC L^ solution 1 and 2 were mixed in a small container. The membrane was placed in this solution for 1 min, then blotted with 3MM paper to remove excess liquid. It is then covered with saran wrap and shielded from the light for 1 min. The membrane is then exposed to pieces of Kodak XAR5 film for 15 seconds upwards until the right exposure is found.