ResultsResults

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

ReferencesReferences

1. Measuring the concentration of protein by BCATM Protein Assay Kit

- Sample : cell lysate, medium2. 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 byWestern 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 MaterialsMethods 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.

ConclusionsConclusions

IntroductionIntroduction

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 Determination of Adiponectin from 3T3-L1 cell by Western Blotby Western Blot

Atittaya Buareaung, Thawankorn Suansong, Pilaiwan SiripurkpongAtittaya Buareaung, Thawankorn Suansong, Pilaiwan SiripurkpongDepartment 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)

BSA Skim milk (oxoid) Skim milk (from Japan)

1st Antibody –1:5,000

Chem. image X-ray film

1st Antibody –1:10,000

Chem. image X-ray film

Figure 4.

1st Antibody –1:15,000

Chem. image X-ray film

Figure 5.2nd Antibody –1:5,000 2nd Antibody –1:10,000

Chem. image X-ray film Chem. image X-ray film

Method Condition

Protein amount 10 µg 20 µg 30 µg 40 µg

Transfer protein90 minw/ ice

90 minw/out ice

180 minw/ ice

-

Blocking reagent BSASkim milk (Oxoid)

Skim milk (Japan)

-

1st Antibody 1:5,000 1:10,000 1:15,000 -

2nd Antibody 1:5,000 1:10,000 - -

Table 1. The optimal conditions for adiponectin detection

AcknowledgementAcknowledgement

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.

ProteinAmount

g g