1. Results 1.Miller JR, Siripurkpong P, Hawes J, Majdalawieh A, Ro HS, McLeod RS. The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARgamma-dependent and PPARgamma-independent mechanisms. J Lipid Res. 2008 Mar;49(3):550-62. 2.Wikipedia. Adiponectin. California. Wikimedia Foundation, Inc.; 2004 [updated 2009 Dec 17; cited 2010 Mar 20]. Available from: http://en.wikipedia.org/wiki/Adiponectin References 1.Measuring the concentration of protein by BCA TM Protein Assay Kit - Sample : cell lysate, medium 2. Separating protein by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) - Marker : Pre-stained molecular weight  Western Blot Protein molecular weight  Comassie blue - Sample : cell lysate, medium 3. Detection of the Adiponectin by Western Blot The conditions for detection of the adiponectin from 3T3-L1 cell by Western blot technique. 1) The loading protein on SDS-PAGE - Protein Amount (cell lysate) : 10 µg, 20 µg, 30 µg, 40 µg 2) Time for transferring protein into nitrocellulose membrane - 90 min w/ ice Vs 90 min w/o ice - 90 min w/ ice Vs 180 min w/ ice 3) Type of blocking reagent - BSA, Skim milk (Oxoid), Skim milk (from Japan) 4) Dilution of primary antibody (mouse anti-adiponectin antibody) - 1:5,000 1:10,000 and 1:15,000 5) Dilution of secondary antibody (goat anti-mouse IgG-HRP) - 1:5,000 1:10,000 Methods and Materials These optimal conditions will be effectively useful for study the adiponectin levels from 3T3-L1 cell, both in cell and in medium. However, proteins in the polyacrylamide gel cannot completely transfer into the nitrocellulose membrane, which this problem will need to be solved in the further study. Conclusions Introduction Adiponectin is a protein hormone, secreted from adipose tissue. It plays an important role in the control of glucose and fat metabolism in the body. Therefore, when the lower level of adiponectin occurs, it could be the cause of many diseases. Particularly Coronary Artery disease and Diabetes Mellitus type 2, which is a major health problem of Thailand. Recently, adiponectin is widely studied from many researchers. There are several methods for detection of the adiponectin. Western Blot is considered as one of popular and reasonable method because it has the high sensitivity and specificity, and does not require tools that are very expensive. Therefore, the purpose of this study is to optimize the conditions for detection of the adiponectin from 3T3-L1 cell by Western blot technique. The loading protein on SDS-PAGE, time for transferring protein into nitrocellulose membrane, type of blocking reagent, the primary and secondary antibody dilutions were optimized for adiponectin detection. Determination of Adiponectin from 3T3-L1 cell by Western Blot Atittaya Buareaung, Thawankorn Suansong, Pilaiwan Siripurkpong Atittaya Buareaung, Thawankorn Suansong, Pilaiwan Siripurkpong Department of Medical Technology, Faculty of Allied Health Science Thammasat University Figure 1. Figure 2a. Various amount of protein transfer by using ice for 90 min Figure 2b. Various amount of protein transfer by using ice for 180 min Figure 3. Our results showed that the optimal conditions for adiponectin detection by Western blot are 10 μg of loading protein (Fig.1), 180 minutes for protein transfer with ice surrounding chamber (Fig.2a.&2b.). The proper blocking reagent is Skim Milk from Japan (Fig.3), and the dilution of the appropriate primary is 1:15,000 (Fig.4) and the dilution of the appropriate secondary antibodies is 1:10,000 (Fig.5) BSASkim milk (oxoid)Skim milk (from Japan) 1 st Antibody –1:5,000 Chem. image X-ray film 1 st Antibody –1:10,000 Chem. image X-ray film Figure 4. 1 st Antibody –1:15,000 Chem. image X-ray film Figure 5. 2 nd Antibody –1:5,0002 nd Antibody –1:10,000 Chem. image X-ray film MethodCondition Protein amount10 µg20 µg30 µg40 µg Transfer protein 90 min w/ ice 90 min w/out ice 180 min w/ ice - Blocking reagent BSA Skim milk (Oxoid) Skim milk (Japan) - 1 st Antibody1:5,0001:10,0001:15,000- 2 nd Antibody1:5,0001:10,000-- Table 1. The optimal conditions for adiponectin detection Acknowledgement We are grateful to thank the Thammasat University for research scholarship and Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University for providing the equipments us to conduct this research. Protein Amount gg gg