Bacterial transformation-
BL21 E. coli cells were incubated with pQE60 plasmid designed to express his-tagged ZAG and were inoculated for 5 min in an Eppendorf tube on ice. Media used in this experiment is 2YT which is composed of 1.6% Tryptone (w/v), 1% Yeast extract (w/v) and 0.5% Sodium Chloride (NaCl) (w/v) in distilled water. After incubation samples were heat-shocked at 42°C for 45 sec then returned to ice for 2 mins. Media was added and after a 1h incubation the mixture was spread evenly on Amp plates. The spreader was passed through the Bunsen after each plate to prevent cross contamination. Plates were then covered in parafilm and incubated at 37°C overnight for growth of single colonies. Colony growth results from a successful transformation as the pQE60 plasmid expresses ampicillin resistance.
Miniprep DNA purification method-
Single colonies were taken from the transformed plate and the pipette is dipped in 20mL falcon tubes containing 2YT media and ampicillin. The tubes were kept overnight in shaking incubator at 37°C. These cultures underwent Wizard Plus SV Minipreps DNA purification System (Promega, #TB225). This is carried out by production of clear lysate. The pellet was resuspended and mixed thoroughly in 250µL in Cell resuspension solution along with 250µL of cell lysis solution. Alkaline protease solution of 10µL is added to the mixture and incubated 5 mins. 350µL of Neutralization solution is added and centrifuged at top speed for 10 mins and transferred to Eppendorf tubes. A spin column was placed in a collecting tube, and the cell lysate was deposited into the spin column and centrifuged at top speed for 1 minute at room temperature in a microfuge. The flow through was removed. Add 750µL of wash solution and centrifuge at high speed for 1 min before repeating with 250µL of wash solution This solution is allowed to centrifuge at top speed for 2 min. The solution is transferred to the microcentrifuge tube (1.5mL) and 100µL Nuclease free water is added to it. After centrifugation for 1 min at top speed, the solution which is eluted is DNA plasmid pQE60 which is stored at -20°C.
Restriction Digest Analysis-
Gel preparation for restriction digest analysis is made out of Agarose gel (0.8% agarose powder (w/v), Trichloroacetic acid (TAE buffer)). This gel is microwaved and stirred at regular intervals till it becomes transparent liquid. The liquid is allowed to cool and poured into the gel casket. Ethidium bromide 1.5µL is added to it and mixed until a clear gel is not obtained. A comb was inserted and left until it was set. 5µL of 1 kb DNA ladder and 10µL of each sample were pipetted into the wells, and the gel was run for 30 min at 75 volts. After running the samples, the gel was placed under U.V light to image the bands. Single as well as double digests were carried out from the miniprep samples. The enzymes included were Bam HI, Eco RI, Bgl III and Hind III. The simulated version of this was made up using Snapgene software (version 6.2).
Protein Purification-
Transformed BL21-PQE60 ZAG expressing single colonies were taken from plate using a pipette tip and dispensed into 20 mL falcon tubes with 2YT media and ampicillin (100µg/mL). The resulting culture was then transferred to conical flask containing 400mL of 2YT media and ampicillin grown overnight. The culture was transferred to 500mL drums and spun 3500xg for 20 min at room temperature in worktop centrifuge. This process gives out a resultant pellet. The pellet was combined with lysis solution (50 mM Tris-HCl, pH 8.0, 25% sucrose (w/v), 1 mM EDTA), and cells were lysed with lysozyme (1 mg) dissolved in lysis buffer before being incubated on ice for 30 min. The presence of viscosity is due to the inclusion of lysozyme. The mixture was then incubated on ice for 30 min with MgCl2 (10 mM), MnCl2 (1 mM), and DNase I (10 µg/mL). Viscosity of the solution decreases as DNase I is added. Detergent buffer (0.2 M NaCl, 1% deoxycholic acid (w/v), 1% Nonidet P-40 (v/v), 20 mM Tris-HCl (pH 7.5), 2 mM EDTA) was added to the cell lysate, incubated for 20 min and then centrifuged at top speed for 10 min at 4°C. The supernatant is discarded and five washes of 0.5% Triton X-100, 1 mM EDTA solution. This process after repeating continuously gives us a tighter pellet.
The samples after washing were transferred to Eppendorf tubes of 1.5mL and then can be spun at top speed for 10 mins at 4°C. This is done in order to bring down all the protein into the pellet. The denaturation of the protein is done by 1mL of 8M urea or 1mL of Guanidium Hydrochloride to remove recombinant protein from inclusion bodies and 3 mL of Refolding buffer (0.1 M Tris-HCl, 2mM EDTA, 0.4 M L-arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione) was added and incubated overnight. The first was taking 1mL of the overnight incubation and placing it in vivaspin 20 centrifugal concentrator. Refolding buffer was gradually added to a total of 20 mL. The sample was then spun at 3500xg until 5 mL of concentrated ZAG solution was left in the top part of the vivaspin tube. This sample was then collected and analyzed for protein concentration in Nanodrop (ThermoFisher, 701-058112). The proteins were measured at wavelength of 280nm UV visible spectrum and which enables high speed accuracy of 1.5µL samples. The concentrations are measured in mg/mL. The integral protein structure was made up by using UCSF chimera (version 1.16).
Lipolysis assay-.
It is a colorimetric assay kit (Sigma Aldrich MAK211) that contains synthetic catecholamine isoproterenol that stimulates β adrenergic receptors. This 3T3-L1 Adipocytes were assessed in this assay. The 3T3-L1 preadipocytes are mainly fibroblasts which are grown and differentiated in 12-well cell culture plates in presence of media Fetal Bovine Serum (FBS) (20). Day-10 cells after adipogenic induction were used for the lipolysis assay. After the differentiation, wash the cells two times with 100µL of lipolysis assay buffer. Remove the wash buffer and replace it with 150µL Lipolysis Assay Buffer. Addition of 1.5µL of 10µM isoproterenol which stimulates lipolysis. 20µL of media (cells) is being added into 96 well plate with addition of 30µL of lipolysis assay buffer so that the total reaches to 50µL. The reaction mix of glycerol is being made up by making the total to 50µL with fixed mixture of 46µL of glycerol assay buffer and 2µL each of glycerol probe and glycerol enzyme mix to the 96 well titre plate. The plate is incubated at room temperature for 30 mins protected from light. The absorbance is read at 570nm in a microtiter plate reader.
Oil Red O staining-
Oil red O staining is used to quantify neutral lipid droplets (Sigma Aldrich MAK194). 3T3 L1 adipocytes grown and differentiated on 12 well plates were used, with triplicate determinations for each condition (no ZAG, ZAG, mutant ZAG etc.). After ZAG treatment as indicated on the figures, the media was aspirated and cells were incubated in 10% (v/v) formalin. Cells were washed in 60% isopropanol and then Oil Red O (0.2%) v/v added for 10 minutes. Cells were then washed with water 4 times. 1mL of isopropanol (VWR 67-63-0) was used to elute the stain. 100µL aliquots were added to a 96-well plate and absorbance measured at 540nm. The 12-well cell plate was also imaged with EVOS® FL Auto Imaging System (Leica) to image the stained lipid droplets at 10x or 20x magnification.
Protein analysis by western blotting-
The stand for gel to be placed with glass slides of 1.5mm for the gel to be set. The running gel and stacking gel is made with given ingredients. Tris-glycine SDS-polyacrylamide running gel (1.5M Tris-HCl, pH8.8, 0.1% (v/v), 10% SDS, 30% (v/v) acrylamide mix, 10% (v/v) ammonium persulfate and 0.1% (v/v) TEMED) (Thermofisher,17919) combined with 5% stacking gel; (1.0M Tris-HCl, pH6.8, 0.1% (v/v), 10% SDS, 30% (v/v) acrylamide mix, 10% (v/v) ammonium persulfate and 0.1% (v/v) TEMED). Gels were run in SDS-Running buffer (0.19M glycine, 25mM Tris-HCl, and 0.1% (v/v) SDS) at 150V for 2-h period. Running buffer is put with TEMED in it and brought to level till the top with water. After removing the water, stacking buffer is added with TEMED and wells are inserted quickly. The gels are left untouched till it solidifies and wells are formed. 3µL of pre-stained protein molecular weight Precision Plus Protein™ Standard ladder (Bio-Rad, 1610374) and 10µL of each sample is added to 15 well polyacrylamide gels 10% (w/v) using a 20 µL pipette. Gels were secured in clamp stands and submerged with running buffer comprising of glycine (0.19 M), SDS 0.1% (w/v) and Trizma®base (25 mM). The gel apparatus was then attached to a power source and was set at 100 V for 60 min and then increased to 150 V when samples had run halfway down the gel.
Wet transfer and immunoblotting-
Once the separation was complete, the gel was placed into a transfer cassette against nitrocellulose paper (ThermoFisher, LC2006) via the use of Mini Trans-Blot® Cell apparatus (Bio-Rad Laboratories, California) following manufacturers protocol, supported with sponge and card. The assembled transfer cassette was submerged in transfer buffer consisting of glycine (0.19 M), Trizma®base (25 mM) and methanol 20% (v/v). Protein transfer from the gel to the nitrocellulose was at 60 V for 135 min. The transferred blot is then poured with ponceau dye (0.1% (v/v) Ponceau S, 5% (v/v) acetic acid) (ThermoFisher, A40000279) to visualize the proteins run on the paper. The dye is then washed with PBS. The nitrocellulose paper was incubated in blocking buffer comprising of Bovine Serum Albumin (BSA) 3% (w/v) in TBST (20mM Tris-HCl, 150mM NaCl, 0.1% Tween (v/v) for 1 h. The membrane was then placed in appropriate primary antibody (1:1000) diluted in BSA 1% (w/v), TBST and rotated overnight at 4°C. After primary incubation, blots underwent three 5-minute washes with TBST or PBST whilst rocking. They were then incubated with the corresponding LI-COR IR Dye-conjugated anti-mouse/rabbit secondary antibody (1:10,000) diluted in BSA 3% (w/v) and TBST for 1 h at room temperature (23°C). The antibodies used were mostly rabbit as it gives us a bright green fluorescence. Subsequently, blots underwent another three 5-minute wash steps in TBST. Proteins were visualized using the LI-COR Infrared 9120 imaging system (Odeyssesy®) at 700nm and 800 nm channels following manufacturer’s protocol. Data was analyzed via Image Studio Lite (Cambridge, UK) imaging system.