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1、Protocols(Proteomics Core Facility, Biocenter Oulu, Dept. of Biochemistry, University of Oulu, Finland)Abbreviations1. Sample preparation1.1. Escherichia coli1.2. Saccharomyces cerevisiae1.2.1. Whole intracellular extract1.2.2. Mitochondria1.3. Mouse1.3.1. Kidney1.3.2. Bones (calvaria and femur)1.3.
2、3. T-cells from spleen1.3.4. Fibroblast cell line NIH3T31.4. Human1.4.1. Serum1.4.2. Tooth pulp tissue2. IEF (Isoelectric focusing)2.1. In-gel rehydration 2.2. Sample cup loading 3. SDS-PAGE (SDS-polyacrylamide gel electrophoresis)3.1. Casting the SDS gels3.1.1. 2-D SDS-PAGE3.1.2. 1-D SDS-PAGE3.2. E
3、lectrophoresis3.2.1. 2-D SDS-PAGE3.2.2. 1-D SDS-PAGE4. Protein detection4.1. Silver staining4.2. Western Blotting5. Protein identification (mass spectrometry)6. Troubleshooting7. Links to other protocols8. References9. ChemicalsAbbreviationsAPSBisCHAPSDTTPMSFPonceau SSDSTCEPTEMEDTrisAmmonium Persulf
4、ateN, N-Methylene-bisacrylamide3-(3-cholamidopropyl)dimethyl-ammonio-1-propanesulfonate1,4-dithio-DL-threitolPhenylmethanesulfonyl Fluoride3-Hydroxy-4-(2-sulfo-4-4-sulfophenylazophenylazo)-2,7-naphthalenedisulfonic acid sodium saltSodium dodecyl sulfateTris-(2-carboxyethyl)phosphine hydrochlorideN,N
5、,N,N-TetramethylethylenediamineTris-hydroxymethylaminomethane1. Sample preparation1.1. Escherichia coliThe E. coli strain RB791 was grown in LB medium and cells (50-100 ml) collected at different growth stages. After a centrifugation (10 min, 8000 rpm, 4 C) the cells were washed twice with 2 ml TE-P
6、MSF (1 mM EDTA, 0.1 M Tris, 14 M PMSF, pH 7.5). The pellet was then resuspended in 3 ml TE-PMSF, disrupted by French Press with 900 PSI (62.1 bar) and the cell lysate centrifuged (30 min, 15.300 rpm, 4 C). The supernatant was collected and the protein concentration determined with a commercially ava
7、ilable kit (RotiNanoquant) according to Bradford, 1976. Aliquots of 100 g were dried (SpeedVac) and stored at - 20 C.Prior to separation in the first dimension (IEF) the proteins were resuspended in 350 l urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3
8、-10, 100 mM Tris/HCl, pH 7.5, 1 mM EDTA, 14 M PMSF) and transfered by in-gel rehydration into the IEF gels.1.2. Saccharomyces cerevisiae1.2.1. Whole intracellular extractFor the analysis of the fermentative growth the S. cerevisiae wild type strain BJ1991 was cultivated on rich YPD (1% (w/v) yeast e
9、xtract, 2% (w/v) peptone, and 2% (w/v) D-glucose) and collected at an optical density (600 nm) of 1.1. For the study of the respiration a wild type culture was grown in YPD to an optical density (600 nm) of 1-2, then pelleted, and transferred to YPG containing 3% (v/v) glycerol instead of D-glucose.
10、 After 16 h cells were collected, centrifuged (10 min, 8000 rpm, 4 C) and washed twice with 2 ml TE-PMSF (1 mM EDTA, 0.1 M Tris, 14 M PMSF, pH 7.5). Then the pellet was resuspended in 3 ml TE-PMSF and disrupted by French Press with 900 PSI (62.1 bar). The cell lysate was centrifugated (30 min, 15.30
11、0 rpm, 4 C) and the supernatant collected. The protein concentration was determined with a commercially available kit (RotiNanoquant) according to Bradford, 1976. Aliquots of 100 g were dried (SpeedVac) and stored at - 20 C.Prior to separation in the first dimension (IEF) the proteins were resuspend
12、ed in 350 l urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris/HCl, pH 7.5, 1 mM EDTA, 14 M PMSF) and transfered by in-gel rehydration into the IEF gels.1.2.2. MitochondriaYeast cells were grown and collected as described above. The mitoch
13、ondria were isolated according to Meisinger et al., 2000. In brief, cells were pelleted by centrifugation (3.000 g, 5 min), washed with distilled water and resuspended under slowly shaking for 20 min at 30 C in 2ml/ g DTT buffer (100 mM Tris-H2SO4 pH 9.4, 10 mM DTT). After washing with zymolyase buf
14、fer (1.2 M sorbitol; 20 mM potassium phosphate, pH 7.4) cells were incubated with 5mg/ g (w/v) Zymolyase-20T (Seikagaku Kogyo Co.) in 7 ml/ g zymolyase buffer for 45 min at 30 C for conversation into spheroblasts. Homogenization was carried out by 15 strokes in a glass-Teflon potter in 6.5 ml/ g ice
15、-cold homogenisation buffer (0.6 M sorbitol; 10 mM Tris-HCl, pH 7.4; 1 mM EDTA, 1 mM PMSF; 0.2 % (w/v) bovine serum albumin). This homogenate was diluted with 1 vol ice-cold homogenisation buffer and cell debris and nuclei removed by centrifugation (1500 g, 5 min, 4 C). The supernatant was again cen
16、trifuged (3000 g, 5 min, 4 C) and the mitochondria in the supernatant pelleted by additional centrifugation (12.000 g, 15 min, 4 C). The pellet was washed with SEM (250 mM sucrose; 1 mM EDTA; 10 mM MOPS, pH 7.2), again centrifuged (12.000 g, 15 min, 4 C) and resuspended in SEM to give a final concen
17、tration of 5 mg/ ml. The mitochondrial fraction was treated by 10 strokes in a glass-Teflon potter and loaded onto a three-step sucrose gradient (1.5 ml 60 %, 4 ml 32 %, 1.5 ml 23 %, 1.5 ml 15 % (w/v) sucrose in EM buffer (10 mM MOPS, pH 7.2; 1 mM EDTA). After centrifugation (134.000 g, 1 h, 2 C) th
18、e purified mitochondria were recovered from the 60 %/ 32 % interface, diluted with 2 vol SEM and centrifuged (10.000g, 2 C). The mitochondrial pellet was resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM E
19、DTA, 14 M PMSF) and the protein concentration determined. Protein aliquots (100 g) were stored at -20 C.Prior to separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 l and the proteins transfered by in-gel rehydration into the IEF gels.1.3. Mouse1.3.1. KidneyKidn
20、eys of adult mice (14.5 days) were dissected, washed in 5 vol of ice-cold 20 mM Tris/HCl pH 7.8, 10 mM EDTA, 2 mM EGTA, 2 mM DTT, dried (SpeedVac) and resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA
21、, 14 M PMSF). The protein concentration was determined and aliquots of 100 g stored at - 20 C.Prior to separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 l and the proteins transfered by in-gel rehydration into the IEF gels.1.3.2. Bones (calvaria and femur)Bone
22、s (calvaria and femur) from 3-4 week old mice were collected, freezed with liquid nitrogen and grinded with a pestle to a fine powder. Proteins were precipitated with acetone (80 % acetone, 0.12 % (w/v) DTT) for at least 2 h at 20 C, pelleted by centrifugation (30 min, 14.000 rpm, 4 C) and resuspend
23、ed in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 M PMSF). The protein concentration was determined and aliquots of 100 g stored at - 20 C.Prior to separation in the first dimension (IEF) the IEF gels were
24、 incubated in 350 l urea buffer without protein. Prior separation the protein solution was applied into sample cups placed at the anodic end of the IEF gel.1.3.3. T-cells from spleenSpleen tissue was homogenized, lymphocytes (B- and T-cells) were isolated using a lymphoprep-gradient centrifugation.
25、The purified lymphocytes were added to a T cell purification column and the T-cells collected and centrifuged. The pellet was resuspended in urea buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 1 % (w/v) DTT, 2 % (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 M PMSF) according
26、 to Grg et al., 2000. The protein concentration was determined and aliquots of 100 g stored at - 20 C. For the separation in the first dimension (IEF) the IEF gels were incubated in 350 l rehydration solution (6 M urea, 2 M thiourea, 1% (w/v) CHAPS, 0.4 % (w/v) DTT, 0.5 % (v/v) carrier ampholytes 3-
27、10) according to Grg et al., 2000 without protein. Prior to separation in the first dimension (IEF) the protein solution was applied into sample cups placed at the anodic end of the IEF gel.1.3.4. Fibroblast cell line NIH3T3A culture of a mouse fibroblast derived cell line (NIH3T3) was grown for 48
28、h in Dulbeccos modified Eagle medium (DMEM) containing 3 % fetal calf serum (FCS). Cells were rinsed once with DMEM without FCS and collected from the plates (10 cm) by carefully scratching. Proteins were precipitated with acetone (80 % acetone, 0.12 % (w/v) DTT) for at least 2 h at 20 C, pelleted b
29、y centrifugation (30 min, 14.000 rpm, 4 C) and resuspended in urea buffer (8 M urea, 2 M thiourea, 1 % (w/v) CHAPS, 20 mM DTT, 0.8 % (v/v) carrier ampholytes 3-10, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 14 M PMSF). The protein concentration was determined and aliquots of 100 g stored at - 20 C.For the
30、separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 l and the proteins transfered by in-gel rehydration into the IEF gels.1.4. Human1.4.1. SerumThe serum was collected, proteins centrifuged down and washed in 1 ml TE-PMSF (1 mM EDTA, 0.1 M Tris, 14 M PMSF, pH 7.
31、5). Then the protein pellet was precipitated in acetone (80 % (v/v) acetone, 0.12 % (w/v) DTT) for at least 2 h at 20 C, pelleted by centrifugation (30 min, 14.000 rpm, 4 C) and resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10). The pr
32、otein concentration was determined and aliquots of 100 g stored at - 20 C.Prior to separation in the first dimension (IEF) the volume of the urea buffer was adjusted to 350 l and the proteins transfered by in-gel rehydration into the IEF gels.1.4.2. Tooth pulp tissueIntact and carious (advanced enam
33、el or early to moderate dentinal caries) third molars were removed. External surfaces of the teeth were first cleaned with 75 % (v/v) ethanol and then mechanically to remove all the external soft tissue remnants, blood and cementum. A 2-3 mm deep groove was cut around the teeth with a 0.1 mm one-sid
34、ed diamond disc, avoiding the exposure of the pulp. The cut was placed 3-4 mm apically from the cement-enamel junction in order to preserve as much of the pulp tissue coronally from the cutting line as possible, to maximize the tissue sample size of the tooth. The root was fractured off trough the c
35、utting line with the sharp-edged pliers, and pulp was gently removed from the coronal pulp chamber, washed thrice with 1 x PBS. Total RNA was isolated according to the Trizol protocol (GIBCO-BRL, Gaithersburg, MD, USA) and the proteins sedimented from the organic phase of the extract using isopropan
36、ol.a. Protein separation for silver stainingThe pellet was resuspended in urea buffer (8 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10) and the protein concentration determined. Aliquots of 100 g or 500 g were stored at - 20 C. For the separation in the first di
37、mension (IEF) the volume of the urea buffer was adjusted to 350 l and the proteins transferred by in-gel rehydration into the IEF gels.b. Separation of Cy3 labelled proteinsFor protein labelling with the fluorescence dye Cy3 the protein pellet was first precipitated with 80 % (v/v) acetone for at le
38、ast 2 h at 20 C and pelleted by centrifugation (30 min, 14.000 rpm, 4 C). The following steps were performed according to the manufacturers protocol (CyDye DIGE Fluor Labelling Kit for Scarce Samples, A. Biosciences) with slight modifications. In brief, the protein pellet was resuspended in urea buf
39、fer (8 M urea, 2 M thiourea, 4% (w/v) CHAPS, complete Mini protease inhibitor, pH 8.0), sonicated (10 min, ultrasonic bath) and mixed. After centrifugation (10 min, 11.400 rpm, room temperature) the supernatant was transferred into a new tube and the pH and protein concentration determined. If neces
40、sary the pH was adjusted to 8.0. Protein aliquots of 10 g were adjusted to a final volume of 9 l with urea buffer. For the labelling 2 l (4 mM) TCEP were added, mixed and incubated for 1 h at 37 C in the dark. Now 4 l Cy3 dye (8 nmol) were added, mixed and incubated for 30 min at 37 C in the dark. A
41、fter that 15 l urea buffer containing also 130 mM DTT and 2 % (v/v) carrier ampholytes 3-10 were added and mixed to stop the labelling reaction. The protein extracts were stored at -20 C for later use.For the separation in the first dimension (IEF) the IEF gels were incubated in 350 l urea buffer (8
42、 M urea, 2 M thiourea, 1% (w/v) CHAPS, 20 mM DTT, 0.8% (v/v) carrier ampholytes 3-10) without protein. Prior separation the protein solution was applied into sample cups placed at the anodic end of the IEF gel.For the separation by SDS-PAGE the IEF gels were equilibrated for 15 min in equilibration
43、solution C (100 ml solution contained 10 ml 1 M Tris/HCl pH 8.0, 36 g urea, 30 ml glycerin, 2 g SDS, 0.5 g DTT, few bromphenol blue) instead of equilibration solution A and B.2. IEF (Isoelectric focusing)The IEF is an electrophoretic method for the separation of proteins according to their charge in
44、 a specific pH gradient. Proteins are separated within an electrical field until they reach the position in the gel were their own charge is zero (isoelectric point, pI).2.1. In-gel rehydrationThe protein solution was adjusted with rehydration solution to a final volume of 350 l (for 18 cm long IEF
45、gels). The IPG strips (e.g. pH 310, non linear, pH 4-7) were incubated over night in the rehydration solution to mediate the protein transfer. IEF was carried out at 20 C with the Multiphor II system (Amersham Biosciences) under mineral oil (PlusOne Immobiline DryStrip Cover Fluid, Amersham Bioscien
46、ces) for 55 kVh (500 V 500Vh, 500 V 2500 Vh, 3500 V 10 kVh, 3500 V 42 kVh; linear gradient) according to Buttner et al., 2001.2.2. Sample cup loadingThe IEF gels (18 cm) were incubated over night in 350 l rehydration solution. Prior the separation the sample cups were placed onto the gel surface at
47、the anodic side of the gel. The gels were overlaid with mineral oil (PlusOne Immobiline DryStrip Cover Fluid) to control the correct positioning of the sample cups. Then the protein solution (20-100 l) was applied into the cups. IEF was carried out at 20 C with the Multiphor II system (Amersham Bios
48、ciences) for 52 kVh (150 V 2 h, 300 V 4 h, 1500 V 14 h, 3500 V 8,5h; step gradient) according to Grg et al., 2000. The strips were stored in aluminium foil at - 20C or prepared for SDS-PAGE.3. SDS-PAGE (SDS-polyacrylamide gel electrophoresis)During SDS-PAGE the proteins are separated according to their molecular mass in a polyacrylamide gel of specific concentration. Prior to SDS-PAGE the proteins are saturated with the strong anionic detergent SDS in order to mask the own charge of the proteins, which might disturb the separation. SDS for
