脂肪组织蛋白质提取方法
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几篇文献中脂肪组织蛋白质提取方法如下,与各位感兴趣的朋友分享
1）Proteomics 2004, 4, 438–441
Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide
Samples (from ten unaffected women) were cut into small pieces and frozen at 2757C until needed. Homogenization was performed using a Polytron PT-1200C (Kinematica, Luzern, Switzerland) directly in the lysis buffer (8.4 M urea, 2.4 M thiourea, 5% CHAPS, 50 mM DTT and 1% IPG buffer 3–10 (total volume 2 mL/g fat pad)). Rabilloud et al. [8] reported that thiourea in combination with urea increases the solubility of proteins in IPGs and increases the number of proteins visualized in the second dimension. To improve lysis and protein solubilization, the suspension was gently shaken for 60 min at room temperature, then centrifuged at 70 000 rpm for 1.5 h at 190C using a TL-100 ultracentrifuge (Beckman, Palo Alto, CA, USA). When attempting to solubilize and separate proteins from abdominal omental adipose tissue, some difficulties arose due to the presence of large amounts (often . 50% of the tissue volume) of triglycerides. To circumvent this difficulty, the soluble infranatant, below the fat supernatant, was carefully recovered, avoiding the unhomogenized material at the bottom of the centrifuge tube. The protein concentration in the collected fraction was measured according to the Bradford dye-binding procedure [9]. The original protein extract was diluted in rehydration solution containing 8 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer, 0.002% bromophenol blue and 10 mM DTT (depending on the gel 50 mM DTT or 100 mM hydroxyethyl disulfide (HED) was sometimes used in place of 10 mM DTT) before IEF. For IPG 6–11, 100 mg of protein diluted in 100 mL of rehydration solution was applied by cup-loading close to the anode [1]. For IPG 3–10, 80 mg of protein was applied by in-gel sample rehydration [10, 11] in 350 mL of the above solution. The experiments were performed in duplicate for each condition and pH range. The strips (18 cm) were focused at 0.05 mA/IPG strip in the IPGphor IEF System (Amersham Biosciences, Uppsala, Sweden). The running conditions for IPG 6–11 were: 0–300 V in 1 min, 300 V for 3 h, from 300–600 V in 2 h, from 600–1000 V in 2 h, from 1000–8000 V in 3 h, and 8000 V for 5 h. The running conditions for IPG strips 3–10 were those reported by Görg et al. [1], with minor modifications, including an additional step at 120 V for 1 h, and a 90 min ramp from 1000–8000 V, instead of a 30 min ramp. The step at 8000 V was maintained until 35 kVh was reached. After IEF, the strips (pH 6–11 and 3–10) were equilibrated and SDSPAGE was performed using a Protean II XL system (Bio- Rad Laboratories, Hercules, CA, USA). Gels (12.5%) in a 18undefined20undefined1 mm format were fixed overnight and silver stained.
2）IDENTIFICATION OF THE ANTI-OBESITY AGENT SODIUM TUNGSTATE TARGETS IN WHITE ADIPOSE TISSUE FROM OBESE RATS
S. Barceló, H. Corominola, M. Claret, M. Julià, and R. Gomis
Endocrinology and Diabetes Unit, Laboratory of Experimental Diabetes, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, C/ Villarroel 170, Barcelona 08036 sbarcelo@clinic.ub.es
Methods. Sodium tungstate (2 mg/ml in drinking water) was administered during 32 days to obese insulin resistant Wistar rats, (obesity was induced by feeding the animals with a “cafeteria” diet during 30 days). Epidydimal WAT from treated and untreated obese and lean animals was homogenised in a buffer containing urea (7 M), thiourea (2 M), CHAPS (2% w/v), DTT (65 mM), ampholines (0.5 % v/v), orthovanadate (10mM) and protease inhibitors. Proteins were separated by two-dimensional electrophoresis (2D-PAGE) and visualised by silver staining. Differential expression of proteins was analysed using Melanie software. Trypsin digests were analysed by MALDI-TOF and proteins identified by peptide mass-fingerprinting.
3) Proteomics 2001, 1, 819–828 Thiourea enhances mapping of the proteome
from murine white adipose tissue
White adipose tissue (WAT), epididymal fat pads, from obese and diabetic ob/ob mice (Umeå strain, M&B, Ry, Denmark) were frozen directly after dissection and stored at –150_C until homogenisation. Before homogenization the fat pads were weighed and thawed in the same buffer as used for homogenisation (9 M urea, 30 mM DTT, and 0.5% IPG buffer). The homogenisation (total volume 2 mL/g fat pad) was performed at room temperature, with a glass-teflon homogeniser, 400 rpm, 8 strokes, followed by centrifugation at 350 000 g (average), 19_C, for 30 min (Optima TLX ultracentrifuge and rotor TLA 120.1 from Beckman Instruments, Palo Alto, CA, USA). Four fractions were obtained; a supernatant consisting of fat; a thin interphase below the fat (peeled off with a pair of tweezers); a water soluble infranatant which was collected while avoiding a small amount of unhomogenised material at the bottom of the centrifuge tube. The infranatant was aliquoted and stored at –80_C until the 2-D analysis was performed. A crude estimate of the protein concentration was obtained by 1-D SDS-PAGE analysis with bovine serum albumin and cytochrome c as standards (Gelcodestaining, according to the manufacturer’s instructions). Tris-glycine gels and XCell II Mini- Cell were from Novex (San Diego, CA, USA). To the analytical 2-D gels 25 _g protein was loaded corresponding to approx. 20 mg wet adipose tissue. Liver tissue from ob/ob mice were homogenised in the same manner and in the same buffer (9 M urea, 30 mM DTT, and 0.5% IPG buffer) as the fat pads. The homogenized liver samples were divided into two parts and fat from the epididymal fat pads (see above) was added to one of them, (1 mL fat/1 mL of liver homogenate). All liver samples, both with and without fat added, were homogenised again (glass-teflon homogeniser, 400 rpm, 8 strokes) and centrifuged at 350 000 g (average), 19_C, for 30 min. The aqueous phase of each sample was collected.
4) sample preparation (analytical gel): (from internet)
WAT (White adipose tissue): Sixteen mg of dried white adipose tissue was mixed with 60 µl of a solution containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of bromophenol blue. The whole final diluted sample (150 µg) was loaded in a cup at the cathodic end of the IPG gels。
5）上海医学2003年第26卷第一期
肥胖与非肥胖者腹部皮下及内脏脂肪组织蛋白质组差异的四例研究
凌雁 高鑫 陆志强
脂肪组织蛋白质的提取:将脂肪组织样品置于玻璃匀浆器中,按每毫克样品2 μl 裂解液的比例分别加入相应体积的裂解液(9 mol/ L 尿素,4 %CHAPS ,65 mmol/ L DTT) ,冰浴中用玻璃匀浆器上下匀浆共15 次。将匀浆液置于eppendorf 管中,4 ℃、15 000 r/ min 离心1 h 后吸取少量上清液,用Bradford 法测定总蛋白质浓度, 其余上清液于- 78 ℃冻存备用。
1）Proteomics 2004, 4, 438–441
Improved resolution of the human adipose tissue proteome at alkaline and wide range pH by the addition of hydroxyethyl disulfide
Samples (from ten unaffected women) were cut into small pieces and frozen at 2757C until needed. Homogenization was performed using a Polytron PT-1200C (Kinematica, Luzern, Switzerland) directly in the lysis buffer (8.4 M urea, 2.4 M thiourea, 5% CHAPS, 50 mM DTT and 1% IPG buffer 3–10 (total volume 2 mL/g fat pad)). Rabilloud et al. [8] reported that thiourea in combination with urea increases the solubility of proteins in IPGs and increases the number of proteins visualized in the second dimension. To improve lysis and protein solubilization, the suspension was gently shaken for 60 min at room temperature, then centrifuged at 70 000 rpm for 1.5 h at 190C using a TL-100 ultracentrifuge (Beckman, Palo Alto, CA, USA). When attempting to solubilize and separate proteins from abdominal omental adipose tissue, some difficulties arose due to the presence of large amounts (often . 50% of the tissue volume) of triglycerides. To circumvent this difficulty, the soluble infranatant, below the fat supernatant, was carefully recovered, avoiding the unhomogenized material at the bottom of the centrifuge tube. The protein concentration in the collected fraction was measured according to the Bradford dye-binding procedure [9]. The original protein extract was diluted in rehydration solution containing 8 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer, 0.002% bromophenol blue and 10 mM DTT (depending on the gel 50 mM DTT or 100 mM hydroxyethyl disulfide (HED) was sometimes used in place of 10 mM DTT) before IEF. For IPG 6–11, 100 mg of protein diluted in 100 mL of rehydration solution was applied by cup-loading close to the anode [1]. For IPG 3–10, 80 mg of protein was applied by in-gel sample rehydration [10, 11] in 350 mL of the above solution. The experiments were performed in duplicate for each condition and pH range. The strips (18 cm) were focused at 0.05 mA/IPG strip in the IPGphor IEF System (Amersham Biosciences, Uppsala, Sweden). The running conditions for IPG 6–11 were: 0–300 V in 1 min, 300 V for 3 h, from 300–600 V in 2 h, from 600–1000 V in 2 h, from 1000–8000 V in 3 h, and 8000 V for 5 h. The running conditions for IPG strips 3–10 were those reported by Görg et al. [1], with minor modifications, including an additional step at 120 V for 1 h, and a 90 min ramp from 1000–8000 V, instead of a 30 min ramp. The step at 8000 V was maintained until 35 kVh was reached. After IEF, the strips (pH 6–11 and 3–10) were equilibrated and SDSPAGE was performed using a Protean II XL system (Bio- Rad Laboratories, Hercules, CA, USA). Gels (12.5%) in a 18undefined20undefined1 mm format were fixed overnight and silver stained.
2）IDENTIFICATION OF THE ANTI-OBESITY AGENT SODIUM TUNGSTATE TARGETS IN WHITE ADIPOSE TISSUE FROM OBESE RATS
S. Barceló, H. Corominola, M. Claret, M. Julià, and R. Gomis
Endocrinology and Diabetes Unit, Laboratory of Experimental Diabetes, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, C/ Villarroel 170, Barcelona 08036 sbarcelo@clinic.ub.es
Methods. Sodium tungstate (2 mg/ml in drinking water) was administered during 32 days to obese insulin resistant Wistar rats, (obesity was induced by feeding the animals with a “cafeteria” diet during 30 days). Epidydimal WAT from treated and untreated obese and lean animals was homogenised in a buffer containing urea (7 M), thiourea (2 M), CHAPS (2% w/v), DTT (65 mM), ampholines (0.5 % v/v), orthovanadate (10mM) and protease inhibitors. Proteins were separated by two-dimensional electrophoresis (2D-PAGE) and visualised by silver staining. Differential expression of proteins was analysed using Melanie software. Trypsin digests were analysed by MALDI-TOF and proteins identified by peptide mass-fingerprinting.
3) Proteomics 2001, 1, 819–828 Thiourea enhances mapping of the proteome
from murine white adipose tissue
White adipose tissue (WAT), epididymal fat pads, from obese and diabetic ob/ob mice (Umeå strain, M&B, Ry, Denmark) were frozen directly after dissection and stored at –150_C until homogenisation. Before homogenization the fat pads were weighed and thawed in the same buffer as used for homogenisation (9 M urea, 30 mM DTT, and 0.5% IPG buffer). The homogenisation (total volume 2 mL/g fat pad) was performed at room temperature, with a glass-teflon homogeniser, 400 rpm, 8 strokes, followed by centrifugation at 350 000 g (average), 19_C, for 30 min (Optima TLX ultracentrifuge and rotor TLA 120.1 from Beckman Instruments, Palo Alto, CA, USA). Four fractions were obtained; a supernatant consisting of fat; a thin interphase below the fat (peeled off with a pair of tweezers); a water soluble infranatant which was collected while avoiding a small amount of unhomogenised material at the bottom of the centrifuge tube. The infranatant was aliquoted and stored at –80_C until the 2-D analysis was performed. A crude estimate of the protein concentration was obtained by 1-D SDS-PAGE analysis with bovine serum albumin and cytochrome c as standards (Gelcodestaining, according to the manufacturer’s instructions). Tris-glycine gels and XCell II Mini- Cell were from Novex (San Diego, CA, USA). To the analytical 2-D gels 25 _g protein was loaded corresponding to approx. 20 mg wet adipose tissue. Liver tissue from ob/ob mice were homogenised in the same manner and in the same buffer (9 M urea, 30 mM DTT, and 0.5% IPG buffer) as the fat pads. The homogenized liver samples were divided into two parts and fat from the epididymal fat pads (see above) was added to one of them, (1 mL fat/1 mL of liver homogenate). All liver samples, both with and without fat added, were homogenised again (glass-teflon homogeniser, 400 rpm, 8 strokes) and centrifuged at 350 000 g (average), 19_C, for 30 min. The aqueous phase of each sample was collected.
4) sample preparation (analytical gel): (from internet)
WAT (White adipose tissue): Sixteen mg of dried white adipose tissue was mixed with 60 µl of a solution containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of bromophenol blue. The whole final diluted sample (150 µg) was loaded in a cup at the cathodic end of the IPG gels。
5）上海医学2003年第26卷第一期
肥胖与非肥胖者腹部皮下及内脏脂肪组织蛋白质组差异的四例研究
凌雁 高鑫 陆志强
脂肪组织蛋白质的提取:将脂肪组织样品置于玻璃匀浆器中,按每毫克样品2 μl 裂解液的比例分别加入相应体积的裂解液(9 mol/ L 尿素,4 %CHAPS ,65 mmol/ L DTT) ,冰浴中用玻璃匀浆器上下匀浆共15 次。将匀浆液置于eppendorf 管中,4 ℃、15 000 r/ min 离心1 h 后吸取少量上清液,用Bradford 法测定总蛋白质浓度, 其余上清液于- 78 ℃冻存备用。
