Materials and Reagents

1. Safe-Lock tubes (2.0 ml, Eppendorf, catalog number: 0 22363352 ; 1.5 ml, Eppendorf, catalog number: 022363204)
2. Pipette tips (Rainin, catalog number: 17005873 , 20 μl; catalog number: 17005875, 250 μl; catalog number: 17007090, 1,000 μl)
3. Stainless Steel Beads, 5 mm (Qiagen, catalog number: 69989 ), store at room temperature
4. Tris Hydrochloride (HCl), 1 mol/L solutions (pH = 8.0) (Fisher Scientific, catalog number: BP1758-500 ), store at room temperature
5. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 ), store at room temperature
6. Sodium Chloride (NaCl) (Fisher Scientific, catalog number: S271-10 ), store at room temperature
7. Triton X-100 (Integra Chemical Company, catalog number: T756.30.30 ), store at room temperature
8. cOmplete Mini, EDTA-free protease inhibitor cocktail tablets (Roche, catalog number: 11697498001 ), store at 2-8 °C
9. Optional: phosphatase inhibitors if phosphorylated proteins will be detected (Phosphatase Inhibitor Cocktail 2: Sigma-Aldrich, catalog number: P5726 ; Phosphatase Inhibitor Cocktail 3: Sigma-Aldrich, catalog number: P0044 ), store at -20 °C
10. Pierce ^TM BCA Protein Assay Kit (Thermo Scientific, catalog number: 23225 ), store at room temperature
11. Liquid nitrogen
12. RIPA buffer (see Recipes)
    

Equipment

1. Qiagen TissueLyser II homogenizer (Qiagen, catalog number: 85300 )
2. Thermo Scientific microcentrifuge (refrigerated) (Thermo Scientific, model: Sorvall^TM Legend^TM Micro 17R, catalog number: 75002404 )
   

Procedure

1. Mouse adipose tissue harvesting
   Harvest adipose tissues [including inguinal/subcutaneous white fat pads (sWAT), visceral/epididymal white fat pads (eWAT), interscapular brown fat pads (BAT), etc., as shown in Figure 1, details see article (Mann et al., 2014)] and snap freeze in liquid nitrogen.
   
   
   Figure 1. Mouse fat pads harvested in different locations. A. Subcutaneous WAT (sWAT). B. Interscapular brown AT (BAT). C. Epidydimal WAT (eWAT).
   
   
   
2. Tissue homogenizing
   1. Add 0.5 ml RIPA buffer without Triton X-100 (~100 mg adipose tissue in a 2.0-ml tube with one stainless steel bead) with protease inhibitor and homogenize using the TissueLyser II (Qiagen) at the highest frequency for 3-5 min until clear. Keep on ice.
   2. Spin down at 6,000 x g for 15 min at 4 °C.
   3. Carefully remove the fat cake (refers to the white lipid layer on top of the aqueous layer as shown in Figure 2) and resuspend the loose pellet.
      Note: Alternatively, use the pipette tip to penetrate the fat cake and transfer the solution and the pellet to a new 1.5-ml tube.
      
      
      Figure 2. Fat cakes in different samples. After homogenization and centrifugation, fat cakes, indicated by arrows, are the white lipid layers on top of the aqueous layers in sWAT, BAT and eWAT samples.
      
      
3. Tissue lysis and centrifuging
   1. Add Triton X-100 to a final concentration of 1% (v/v), mix well, and keep on ice.
   2. Incubate at 4 °C for 30-60 min.
   3. Centrifuge at 12,000 x g for 15 min at 4 °C.
   4. Remove upper layer of lipid and transfer the supernatant to a new 1.5-ml tube.
   5. Optional but recommended: Repeat Steps C3 and C4 for twice to get rid of maximum amount of lipid.
   6. Store the extracted samples at -80 °C until further BCA concentration measurement (summarized in Table 1).
      
      Table 1. Protein concentration from different fat pads
      
      
      
   7. Perform Western Blotting (see Figure 3) or other applications.
      
      
      Figure 3. Representative Western blot images of protein samples extracted from AT. A total of 15 μg protein in each sample was loaded on an SDS-PAGE gel. Adiponectin and an internal control β-actin in sWAT (left), eWAT (middle) and BAT (right) were probed.
      

Notes

1. An initial volume of RIPA buffer can be added to 0.6 ml/100 mg tissue. With larger volume of lysis buffer, it is easier to remove the lipid layer and thus achieve higher efficiency of depleting lipid.
2. For more representative Western blotting images, please refer to our cited publications (An et al., 2017 and 2019).
3. This protocol is not applicable to specifically enrich adipose tissue membrane proteins.
   

Recipes

1. RIPA buffer
   50 mmol/l Tris-HCl (pH 8.0)
   0.25 mol/l NaCl
   5 mmol/l EDTA
   1% Triton X-100 (v/v)
   

Acknowledgments

This study was supported by US National Institutes of Health (NIH) grants P01-DK088761, R01-DK55758 and R01-DK099110 (P.E.S.). This protocol was derived from the original research paper published in An et al. (2017).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments conducted in the present study were approved by the Institutional Animal Care and Research Advisory Committee at The University of Texas Southwestern Medical Center (APN# 2015-101207).

References

1. An, Y. A., Sun, K., Joffin, N., Zhang, F., Deng, Y., Donze, O., Kusminski, C. M. and Scherer, P. E. (2017). Angiopoietin-2 in white adipose tissue improves metabolic homeostasis through enhanced angiogenesis. Elife 6: e24071.
2. An, Y. A., Crewe, C., Asterholm, I. W., Sun, K., Chen, S., Zhang, F., Shao, M., Funcke, J.-B., Zhang, Z., Straub, L., Yoshino, J., Klein, S., Kusminski, C. M. and Scherer, P. E. (2019). Dysregulation of amyloid precursor protein impairs adipose tissue mitochondrial function and promotes obesity. Nature Metabolism 1(12): 1243-1257.
3. Benabdelkamel, H., Masood, A., Alanazi, I. O. and Alfadda, A. A. (2018). Comparison of protein precipitation methods from adipose tissue using difference gel electrophoresis. Electrophoresis.
4. Diaz Marin, R., Crespo-Garcia, S., Wilson, A. M. and Sapieha, P. (2019). RELi protocol: Optimization for protein extraction from white, brown and beige adipose tissues. MethodsX 6: 918-928.
5. Mann, A., Thompson, A., Robbins, N. and Blomkalns, A. L. (2014). Localization, identification, and excision of murine adipose depots. J Vis Exp (94). Doi: 10.3791/52174.