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'''Running the Gel'''
#Wear protective gloves when handling gels and solutions#Determine amount of sample needed for 60 x µg of protein in each electrophoresis sample#Prepare reducing sampleTurn on heating block to 70°C about 15 minutes before step 6.##Sample x μl##NuPAGE LDS Sample Prepare 1000 ml of 1x NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer (4X20X) 2.5 μlas follows in 1000mL Pyrex Bottle and mix thoroughly, Running Buffer: ##1. NuPAGE® Reducing Agent SDS Running Buffer (10X20X MOPS) 1 μl50 ml ##2. Deionized Water to 6950 ml 3.5 μlTotal Volume 1000 ml ##Total Volume 10 μl ##Notes: For reduced sample, add • Set aside 800 ml of the reducing agent immediately prior to electrophoresis to obtain 1X NuPAGE® SDS Running Buffer for use in the best results. The presence lower (Outer) Buffer Chamber of more glycerol also increases the viscosity XCell SureLock Mini-Cell • 200 ml of 1X NuPAGE® SDS Running for use in the NuPAGE® LDS Sample Upper (Inner) BufferChamber of the XCell SureLock. By bringing the NuPAGE® LDS Sample Buffer to Mini-Cell. • Keep at room temperature (25°C), the buffer is more manageable.#Heat the Prepare reducing sample for denaturing electrophoresis in this order (reduced or non-reducedTotal volume depends on strength of protein) at 70°C for 10 minutes for optimal results.:#Prepare 1000 ml of 1x NuPAGE® SDS Running Buffer using NuPAGE® SDS Running 1. Sample x μl 2. NuPAGE LDS Sample Buffer(20X4X) as follows: Running Buffer:4°C##3. NuPAGE® SDS Running Buffer Reducing Agent (20X MOPS10X) 50 ml4°C ##Deionized Water 950 ml4. Maximum Total Volume 25 μl ##Total Volume 1000 ml*Mix thoroughly and set aside 800 ml of 5. Notes: For reduced sample, add the 1X NuPAGE® SDS Running Buffer for use in the lower (Outer) Buffer Chamber of the XCell SureLock Mini-Cell*Immediately, reducing agent immediately prior to electrophoresis, add 500μ l to obtain the best results. The presence of NuPAGE® Antioxidant to 200 ml more glycerol also increases the viscosity of 1X the NuPAGE® SDS Running for use in LDS Sample Buffer. By bringing the Upper NuPAGE® LDS Sample Buffer to room temperature (Inner25°C) Buffer Chamber of , the XCell SureLockbuffer is more manageable. Mini# Heat the sample for denaturing electrophoresis (reduced or non-Cell. Mix thoroughlyreduced) at 70°C for 10 minutes for optimal results.#Remove the NuPAGE® Gel from the pouch#Rinse the gel cassette with deionized water2x. Peel off the tape from the bottom of the cassette.#In one smooth motion, gently pull the comb out of the cassette#Rinse Fill the sample wells with 1X NuPAGE® SDS Running Buffer. using loading tips to rinse. Invert the gel and shake to remove the buffer2x. Repeat two Load buffer into wells one more timestime to push all bubbles out.#Orient the two gels in the Mini-Cell such that the notched well-side of the cassette faces inwards toward the Buffer Core. Seat the gels on the bottom of the Mini-Cell and lock into place with the Gel Tension Wedge. #If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette#Fill the Upper Buffer Chamber with a small amount of the running buffer to check for tightness of seal. If you detect a leak from Upper to Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.#Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer(200mL). The buffer level must exceed the level of the wells.#Load an appropriate volume of sample at the desired protein concentration onto the gel #Load appropriate protein molecular weight markers#Fill the Lower (outer) Buffer Chamber with 600 ml of 1X NuPAGE® SDS Running Buffer#Run Immediately run the gel at 200 V constant60 mA/gel, max 100 V. Run for 50-55 minutesuntil samples run onto the foot of the gel. (Check for bubbles emerging from solution to see if electrophoresis is running.)# Refer to step 1 and 2 of Western Blotting'''Removing the Gel after Electrophorsis'''#After electrophoresis is complete, shut off the power, disconnect electrodes, and remove gel(s) from the XCell SureLock. Mini-Cell#Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the cassette’s two plates. The notched (well) side of the cassette should face up.#Push down gently on the knife handle to separate the plates. Repeat on each side of the cassette until the plates are completely separated#Carefully remove and discard the top plate, allowing the gel to remain on the bottom (slotted) plate '''Western Blotting'''#Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer Buffer (20X) as followsin 1000mL Pyrex bottle:Transfer Buffer:##1. NuPAGE® Transfer Buffer (20X) 50 mlat room temp ##NuPAGE® Antioxidant 1 ml##2. Methanol (100 ml##mL for 1 gel) (200mL for 2 gels)3. Deionized Water (849 mlmL for 1 gel) (749 mL for 2 gels) ##4. Total Volume 1000 ml5. Put in 4°C#Use about 700 ml of 1X NuPAGE® Transfer Buffer to soak the pads until saturatedduring electrophoresis. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed. # Put 650mL dH20 into 4°C for step 21.#Cut selected transfer membrane and filter paper to the dimensions of the gel or use Novex® pre-cut membrane/filter paper sandwich. Keep track of shiny side of transfer membrane#Pre-wet PVDF Soak the filter paper and transfer membrane for 30 seconds briefly in methanol, ethanol, or isopropanol. Briefly rinse in deionized water, then place in a shallow dish with 50 ml of 1X NuPAGE® 1X NuPAGE® Transfer Buffer for several minutesimmediately prior to use. #Soak Use the filter paper briefly in 1X NuPAGE® Transfer Buffer immediately prior to use.#Use the gel gel immediately following the run. Do not soak the gel in transfer running buffer#After prying opening the gel cassette remove wells with the Gel Knife. May run some transfer buffer over surface to remove air bubbles.#Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the foot of the gel uncovered. Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller#Turn the plate over so the gel and filter paper are facing downwards over a gloved hand parafilm or clean flat surface#Use the Gel Knife to push the foot out of the slot in the plate and the gel will fall peel off#When the gel is on a flat surface, cut the foot off the gel with the gel knife#Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane shiny side down on the gel, ensuring all air bubbles have been removed#Place another pre-soaked anode filter paper on top of the membrane. Remove any trapped air bubbles#Place two soaked blotting pads into the cathode (-) core of the blot module. The cathode core is the deeper of the two cores and the corresponding electrode plate is a darker shade of gray. Carefully pick up the gel membrane assembly and place on blotting pad in the same sequence, such that the gel is closest to the cathode core (see Figure next pagefollow manual)#Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode core. Place the anode (+) core on top of the pads. The gel/membrane assembly should be held securely between the two halves of the blot module ensuring complete contact of all components#Position the gel/membrane assembly and blotting pads in the cathode core of the XCell II. Blot Module to fit horizontally across the bottom of the unit. There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place#Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. The blot module will only fit into the unit one way, so the (+) sign can be seen in the upper left hand corner of the blot module. Properly placed, the inverted gold post on the right hand side of the blot module will fit into the hole next to the upright gold post on the right side of the lower buffer chamber#Place the Gel Tension Wedge so that its vertical face is against the blot module. Lock the Gel Tension Wedge by pulling the lever forward#Fill the blot module with 1X NuPAGE® Transfer Buffer until the gel/membrane assembly is covered in this buffer. Do not fill all the way to the top as this will only generate extra conductivity and heat#Fill the Outer Buffer Chamber with deionized water by pouring approximately 650 ml in the gap between the front of the blot module and the front of the lower buffer chamber. The water level should reach approximately 2 cm from the top of the lower buffer chamber. This serves to dissipate heat produced during the run#Place the lid on top of the unit#With the power turned off, plug the red and black leads into the power supply. Run the transfer at 30 10 Volts with an expected current of 170mA 100mA for 24 hours in 4°C in an ice bucket or for 60 about 75 minutesroom temp
'''Immunoblot Assay'''
#Rinse membrane twice with waterdwater #Block membrane 20min -2hours (depending on protein) at room temperature with agitation in freshly prepared 5% nonfat dry milk albumin (Catalog 20-200) in TBS (TBS-MLK)#Incubate membrane overnight at 4C 4°C with agitation and anti-Adipsinx, diluted 1:200 2000 in freshly prepared TBS-MLK(dilution depends on protein)#Wash membrane twice (5-10min each) with TBS-Tween#Incubate membrane for 1hr at room temperation with agitation in anti-goat-HRP conjugate (1:2000 dilution in TBS) (dilution depends on protein) and Bio-rad precision protein Strep Tactin-HRP (1:30,000 dilution in TBS-Tween binds to protein ladder)#Wash membrane 3x (5-10min each) with TBS-Tween
'''Chemiluminescence Detection'''
#Use Bio-rad Immu-star WesternC Chemiluminescent Kit. Mix 1 6 mL of Detection Reagent 1 and 2 for each transfer membrane (1 mL of each:1), with a pipette. #Pour over transfer membrane and leave on for one minute. #Dry off with Kimwipe#Bring to the dark room and make sure the door is tightly closed (no light seeping through), before beginning imager. Settings:# Exposure Type: Precision#Take out Kodak scientific imaging film and cut in half. Put the other half back in the box. Exposure Time: Auto#Put Kodak film over transfer membrane in cassette and close cassette. Leave for one minute. (NoteSensitivity: Do not expose film to light. Keep lights off until film is in imaging machine) Standard/High#Put film into imaging machine to processMethod: Chemiluminescence#If nothing shows, put the film back into the cassette Adjust brightness and focus >> Capture Image and leave it in for another 30 minutes. Check back to see if results

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