Western blot鉴定背肩胛棕色脂肪黑皮质素4型受体的研究

doi:10.11843/j.issn.0366-6964.2018.04.023

Abstract

Abstract:

The main objective of this study was focused on expression of low abundance protein and details of the experimental procedure detected by Western blot. Brown adipose tissue (BAT) from 6-8 week old C57/BL6 mice were chosen as experimental materials and detected for the expression of Melanocortin-4-receptor (MC4R) using different parameters including pH and content of Sodium Dodecyl Sulfate (SDS) in electrotransfer buffers. The effects of protein processing methods, components and pH of electrotransfer buffers on transfer efficiency were investigated. The results showed that initial voltage and power were significantly related to the content of sodium SDS and pH of the electrotransfer buffers. The concentration of SDS in the transmembrane buffer was inverse to the transfer voltage and power under the constant circulating membrane. The transmembrane voltage and power of buffer in pH 8.0 were significantly higher than those of buffers in pH 7.6 (P<0.01) and pH 8.6 (P<0.001). The expression of relating proteins, especially MC4R, in group of alcohols mixed lysate (20% Methanol+1% Triton+1% SDS+RIPA) was significantly higher than that in the single RIPA group (P<0.05) and Trizol group (P<0.01). The result indicate that adding enhancers (Triton and SDS) to the lysate buffer can promote the efficiency of protein extraction and improve the sensitivity of antibody for proteins. Those procedure may help to understand the details and improve the sensitivity and repeatability of the Western blot.

CLC Number:

Q503

FAN Kui-kui, LI Qiang, YANG Wen-ya, PAN Deng, LI Hai-jun, DU Chen-guang. Identification of the Melanocortin-4-Receptor in Back Shoulder Brown Adipose Tissue by Western Blot[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 841-846.

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References

[1] JIANG Y, HOU X X, GENG Z, et al. Interpretation criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China[J]. Biomed Environ Sci, 2010, 23(5):341-349.
[2] 董燕, 张枫, 梅柱中, 等. 电转移中蛋白质的透膜现象及其对蛋白质印迹结果的影响[J]. 生物化学与生物物理进展, 2002, 29(3):449-453.
DONG Y, ZHANG F, MEI Z Z, et al. Protein blowthrough in electrophoretic transfer and its effects on Western blotting[J]. Progress in Biochemistry and Biophysics, 2002, 29(3):449-453. (in Chinese)
[3] DEGASPERI A, BIRTWISTLE M R, VOLINSKY N, et al. Evaluating strategies to normalise biological replicates of Western blot data[J]. PLoS One, 2014, 9(1):e87293.
[4] EATON S L, ROCHE S L, HURTADO M L, et al. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting[J]. PLoS One, 2013, 8(8):e72457.
[5] GHOSH R, GILDA J E, GOMES A V. The necessity of and strategies for improving confidence in the accuracy of Western blots[J]. Exp Rev Proteomics, 2014, 11(5):549-560.
[6] HEDA G D, OMOTOLA O B, HEDA R P, et al. Effects of reusing gel electrophoresis and electrotransfer buffers on Western blotting[J]. J Biomol Tech, 2016, 27(3):113-118.
[7] 谢岚, 艾华. Western blot方法中不同电流强度对较大分子蛋白质转膜效率的影响[J]. 国际检验医学杂志, 2012, 33(1):42-44.
XIE L, AI H. Effects of different current densities on efficiency of electrotransfer for the large-molecular-weight protein in Western blot assay[J]. International Journal of Laboratory Medicine, 2012, 33(1):42-44. (in Chinese)
[8] PENNA A, CANHALAN M. Western blotting using the Invitrogen NuPage Novex Bis Tris MiniGels[J]. J Vis Exp, 2007(7):264.
[9] ABEYRATHNE P D, LAM J S. Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots[J]. Can J Microbiol, 2007, 53(4):526-532.
[10] COLLINS M A, AN J Y, PELLER D, et al. Total protein is an effective loading control for cerebrospinal fluid Western blots[J]. J Neurosci Methods, 2015, 251:72-82.
[11] CAWRY N X, YANIK T, WORONOWICA A, et al. Obese carboxypeptidase E knockout mice exhibit multiple defects in peptide hormone processing contributing to low bone mineral density[J]. Am J Physiol Endocrinol Metab, 2010, 299(2):E189-E197.
[12] FAULKNER L D, DOWLING A R, STUART R C, et al. Reduced melanocortin production causes sexual dysfunction in male mice with POMC neuronal insulin and leptin insensitivity[J]. Endocrinology, 2015, 156(4):1372-1385.
[13] KURIEN B T, SCOFIELD R H. Western blotting of high and low molecular weight proteins using heat[M]//KURIENR B T, SCOFIELD R H. Western Blotting:Methods and Protocols. New York, NY:Humana Press, 2015:247-255.
[14] JIN S, FURTAW M D, CHEN H X, et al. Multiplexed Western blotting using microchip electrophoresis[J]. Anal Chem, 2016, 88(13):6703-6710.
[15] GARCÍA-BEA A, WALKER M A, HYDE T M, et al. Metabotropic glutamate receptor 3(mGlu3; mGluR3; GRM3) in schizophrenia:Antibody characterisation and a semi-quantitative Western blot study[J]. Schizophr Res, 2016, 177(1-3):18-27.
[16] MORGAN D A, MCDANIEL L N, YIN T, et al. Regulation of glucose tolerance and sympathetic activity by MC4R signaling in the lateral hypothalamus[J]. Diabetes, 2015, 64(6):1976-1987.
[17] 黄萌, 贤明, 史明艳, 等. MC4R基因沉默对牛成纤维细胞内基因表达的影响[J]. 畜牧兽医学报, 2017, 48(3):403-415.
HAUNG M, XIAN M, SHI M Y, et al. Effects of MC4R gene silencing on gene expression in bovine fibroblast cells[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(3):403-415. (in Chinese)
[18] 张菊, 杜立新, 李宏滨, 等. 绵羊MC4R基因的半定量RT-PCR及生物信息学分析[J]. 畜牧兽医学报, 2010, 41(7):804-810.
ZHANG J, DU L X, LI H B, et al. Semi-quantitative RT-PCR and bioinformatics analysis of sheep MC4R gene[J]. Acta Veterinaria et Zootechnica Sinica, 2010, 41(7):804-810. (in Chinese)
[19] TREINDL F, RUPRECHT B, BEITER Y, et al. A bead-based western for high-throughput cellular signal transduction analyses[J]. Nat Commun, 2016, 7:12852.
[20] HEMMATZADEH F, KAZEMIMANESH M. Detection of specific antigens of Newcastle disease virus using an absorbed Western blotting method.[J]. Iran J Vet Res, 2017, 18(2):92-96.
[21] GILDA J E, GHOSH R, CHEAH J X, et al. Western blotting inaccuracies with unverified antibodies:need for a Western blotting minimal reporting standard (WBMRS)[J]. PLoS One, 2015, 10(8):e0135392.
[22] LEIMGRUBER R M, MALONE J P, RADABAUGH M R, et al. Development of improved cell lysis, solubilization and imaging approaches for proteomic analyses[J]. Proteomics, 2002, 2(2):135-144.
[23] BURGHI V, FERNÁNDEZ N C, GÁNDOLA Y B, et al. Validation of commercial mas receptor antibodies for utilization in Western blotting, immunofluorescence and immunohistochemistry studies[J]. PLoS One, 2017, 12(8):e0183278.
[24] SINKALA E, SOLLIER-CHRISTEN E, RENIER C, et al. Profiling protein expression in circulating tumour cells using microfluidic Western blotting[J]. Nat Commun, 2017, 8:14622.
[25] OKITA N, HIGAMI Y, FUKAI F, et al. Modified Western blotting for insulin and other diabetes-associated peptide hormones[J]. Sci Rep, 2017, 7:6949.
[26] MERPHY R M, LAMB G D. Important considerations for protein analyses using antibody based techniques:down-sizing Western blotting up-sizes outcomes[J]. J Physiol, 2013, 591(23):5823-5831.
[27] HEDA G D, OMOTOLA O B, HEDA R P, et al. Effects of reusing gel electrophoresis and electrotransfer buffers on Western blotting[J]. J Biomol Tech, 2016, 27(3):113-118.
[28] MARANGONI A, FOSCHI C, CAPRETTI M G, et al. Contribution of a comparative Western blot method to early postnatal diagnosis of congenital syphilis[J]. Clin Vaccine Immunol, 2016, 23(5):410-416.

Identification of the Melanocortin-4-Receptor in Back Shoulder Brown Adipose Tissue by Western Blot

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