RELi protocol: Optimization for protein extraction from white, brown and beige adipose tissues

Graphical abstract


Specifications
Value of the Protocol The optimization steps presented in this protocol provide a methodological approach to remove excess contaminating lipids and detergents during protein extraction from adipose tissue. This approach increases the quality and quantity of total extracted proteins from adipose tissue. This improved protocol permits for proper and reproducible loading of investigated proteins and housekeeping genes in western blot analysis

Description of protocol
Adipose tissue (AT) is a major metabolic organ and plays key roles in regulating energy balance. It is highly specialized and distinct fat pads distributed throughout the body have biochemically distinct functions. White adipose tissue (WAT), formed of white adipocytes, plays an important role in energy storage and in the secretion of adipokines, hormones and interleukins [1]. Brown adipose tissue (BAT), formed of brown adipocytes, has the primary function of maintaining core temperature by generating heat through thermogenesis [2]. Within WAT, beige adipocytes are generated as a response to external cues. These adipocytes share similar morphologic features to brown adipocytes, allowing them to participate in thermogenesis [3]. Due to its relevance in physiology and disease, adipose tissue is broadly studied in multiple fields and in multiple experimental models. In mice, epididymal adipose tissue is a classical WAT, interscapular adipose tissue is a classical BAT and the inguinal adipose tissue is beige adipose tissue (BgAT) [4,5] (Fig.1). There are currently several available approaches to extract protein from adipose tissue such as the bullet blender method or commercially available adipose tissue extraction kits [6,7]. These methods to process adipose tissue, however, require specialized equipment such as a bullet blender or can be expensive when assessing a large number of samples. At present, there is a lack of a standardized protocol for protein extraction from AT for Western blot analysis. Other general protein tissue extraction protocols and kits require limited specific equipment and are affordable but do not factor in the high content of lipids in ATand require high concentrations of detergents that interfere with protein quantification [8,9]. To overcome these problems, we propose a novel method for mouse AT protein extraction based on a commercial RIPA buffer protocol adapted for Western blot protein analysis. The selection of RIPA buffer is based on its versatility for extracting proteins from diverse cell fractions (e.g. membrane, nuclear, cytoplasmic) and its compatibility with follow-up assays. We compared this new Removal of Excess Lipids (RELi) method of protein extraction from AT to the Cell Signaling Technologies method, henceforth referred to as "CST method". Our optimized protocol provides a simple, affordable and efficient method to extract AT proteins and reduce lipid content. The final outcome is a protocol that is reproducible, provides higher yields and is affordable for ATextraction and subsequent protein analysis.  5.4 With tweezers, carefully grab the furthest right-hand part of the white adipose tissue layer and cut with scissors, from right to left, removing both white and brown fat pads from the scapula. 5.5 Place the excised chunk of tissue (white adipose tissue layer containing the brown adipose tissue pads) in a dissection dish and remove all white adipose tissue (BAT has a darker color than WAT), as depicted in Figs. 1F and 2 .

Required reagents and equipment
6 Place the adipose tissues in a 1.5mL capped tube and keep in dry ice.

Tissue lysis
7 Weight a 1.5 mL capped tube and tare the balance. 8 Cut the adipose tissue while it is still frozen and add 100 mg to the 1.5 mL capped tube.
Note: keep the tube containing the adipose tissue in dry ice until ready to extract all other samples. 9 Prepare necessary volume of RIPA 1X solution (300mL/50mg of adipose tissue are needed per sample) supplemented with protease and phosphatase inhibitor cocktails for a final concentration of 1/500 and 1/100 respectively. 10 Add 300mL of RIPA 1X supplemented with protease and phosphatase inhibitors to the 1.5mL capped tubes and keep on ice. 11 Set the Fastprep FP120 machine's speed to 6.0 m/s and lyse tissue for 30 s. 12 Remove samples from the Fastprep FP120 machine and chill samples on ice for 45 s. 13 Repeat steps 11-12 two more times or until tissue is completely lysed. 14 Incubate samples on ice for at least 1h. 15 If any solid portion of the adipose tissue remains in the 1.5 mL capped tube, remove it with the help of forceps before continuing onto the next steps. 22 Calculate the volume of protein needed to load 50mg of protein for Western blot, add Laemmli buffer and complete to a total volume of 30mL with RIPA buffer 1X or distilled water. 23 Boil samples for 5 min in a heat block at 100 o C.

Extraction validation by Western blot
24 Load 15mL of the protein preparation for every sample on a 12.5% acrylamide gel and migrate at 120 V for 1h. Note: After migration, the gel can be colored with SimplyBlue solution to verify protein loading. Wash the gel 3 times for 5 min with Milli-Q water to remove SDS and buffer salts. Color the gel with SimplyBlue solution for 2h at RT. To obtain a clear background, decolor gel with Milli-Q water during 1h at RT and place a KimTech on top of the gel to absorb the dye. Take a picture of the gel with a digital imaging system (ImageQuant) and quantify total protein loaded per well with Image J.
25 Transfer proteins on a PVDF membrane for 1h 30min at 100V, 4 o C. 26 Note: After transfer, Ponceau S solution can be used to verify protein loading. Wash the membrane twice with TBS + Tween (TBST), for 5 min to remove all Ponceau off the membrane prior to continuing analysis. Block the membrane with 5% non-fat milk diluted in TBST, for 1h at room temperature (RT). 27 Incubate O/N 4 o C with primary antibody or 1h at RT for β-actin primary antibody. 28 Wash membrane 3 times with TBST for 7 min. 29 Incubate the membrane with secondary antibody for 1h at RT. 30 Wash the membrane 3 times with TBST for 7 min. 31 Develop membrane with a digital imaging system (ImageQuant) using the Clarity Western ECL Substrate. 32 Quantify mean intensity of the bands detected with Image J or alternative gel quantification system.

Method validation
The validation of the above-described method was obtained by means of Western blot analysis comparing the RELi protocol to the CST method. WAT, BAT and BgAT were collected from three adult male C57BL/6 mice and proteins were extracted using both methods with RIPA buffer. Protein concentration was evaluated with the BCA method by measuring absorbance (562 nm) with a TECAN infinite M1000 Pro (Table 1). Samples processed with the CST methods show overestimated protein concentrations due to interference from lipid contamination when compared to our optimized protocol.
To validate the interference of lipids in protein quantification, 50 mg of protein were loaded for every tissue sample on 12.5% acrylamide gels, separated by SDS-PAGE electrophoresis and counterstained with SimplyBlue SafeStain. Considering that all lanes were loaded with the same amount of calculated protein, samples processed with our RELi method presented a higher and more consistent amount of proteins when compared to that obtained from the CST method for all analyzed tissues (WAT, BAT and BgAT) (Fig. 3A-D). To assess the outcome of removal of excess lipids during protein extraction, we performed Western blot analysis on the samples extracted with the CST and RELi method for all three adipose tissues. βactin was used as a housekeeping protein to assess the high variability in the loading. Our results indicate that the CST method yielded inferior amounts of protein from WAT and BgAT (Fig. 3E-F) as determined by β-actin expression when compared to our optimized protocol. The amount of β-actin detected in BAT, however, was not improved by our method. This is probably due to the lower levels of lipids in BAT compared to other types of adipose tissue.
To validate the quality and uniformity of adipose tissue proteins, we evaluated intracellular compartments in adipose tissue by Western blot. Markers of cellular fractions included: cytosolic (βactin), stromal vascular fraction (eNOS), mitochondrial (BCL2), lipid droplet (LD) associated proteins (Perilipin A) and nuclear (Histone H3) for all three types of adipose tissue [10]. Our results show that the RELi method yields higher levels of intracellular fractions when compared to the CST method for WAT and certain fractions of BgAT such as cytosol (Fig. 4A-B) as determined by the enhanced expression of the above-mentioned markers. Conversely, for BAT, the RELi method did not show superiority (Fig. 4C). This may be due to initially lower volumes used for tissue lysis. Nevertheless, when compared to other protocols that use TCA precipitation to extract adipose tissue proteins, our RELi method remains a quicker and more reliable technique for adipose tissue extraction for Western blot analysis [10]. A consistent quantification of protein after extraction is fundamental for reproducibility between samples and thus our optimized protocol will help researchers obtain more reliable protein analysis data.