Protein analysis by immunoblotting

Protein analysis by immunoblotting

Lysate collection: 

• Primary chondrocytes were isolated from sterna of newborn mice and cultured in 6 well tissue culture plates (Corning, 3516)
• After appropriate treatment, tissue culture plates were placed on ice
• Media was aspirated gently by tilting plate without touching bottom of dish
• Cells were washed by adding cold PBS while plates remain on ice
• When cells were ready to collect, PBS was aspirated by tilting plate and ensuring there was little PBS remaining that would dilute the sample
• 100 μl of 1x Cell lysis buffer (CST #9803) with protease and phosphate inhibitor (Thermo Fisher #78440) was added to wells
• Cells were scraped using flat cell lifter to scrape the entire bottom of the well
• Cell lysate will now appear clumpy and will require homogenization
• Pipette the liquid up and down using a p100 pipette without aspirating air to cause bubble formation
• Lysate is then transferred to 1.5 mL microcentrifuge tube 
  • For improved lysis, samples can be freeze thawed once by storing at -80 until frozen and allowed to thaw on ice
• Centrifuge the samples at 10000 rpm on micro centrifuge for 1 minute
• Aspirate lysate without collecting pellet and store at -20 degrees celsius or -80 celsius until ready to use

Sample preparation and loading: 

• Thaw samples on ice
• Measure protein concentration in samples. We utilized Pierce BCA protein assay kit (ThermoFisher, 23225)
• Transfer around 25-50 μg of protein lysates to microcentrifuge tube (just keep amount of protein loaded consistent amongst the samples loaded)
  • Amount of protein loaded will depend upon the protein of interest
• Add 4x LDS (Fisher B0007) to samples to bring final concentration to 1x
• Add 10x Sample reducing agent (Fisher B0009) to final concentration of 1x
• Heat samples at 70 degrees celsius for 10 minutes
• Load samples onto pre-cast Bolt Bis-Tris plus gels (We generally utilize 4-12% gels)

Running gel and transfer: 

• Gels were run using 1x Bolt MES running buffer (Fisher B0002) diluted from 20x stock solution with distilled water
• Gels were run at 200V for 30 minutes
• Gels were transferred utilizing the iBlot dry gel transfer system (ThermoFisher IB21001) 
• Transfer was performed according to the protocol using the 7 minute transfer method

Immunoblotting:

• After transfer, excess membrane was cut. Gloves were utilized when handling membranes while ensuring not to touch area of membrane that proteins were transferred onto
• Membrane was placed in small black box (to prevent light from bleaching secondary antibody) approximately the size of the membrane and 5% BSA solution was added. BSA was dissolved in 1x PBS with 0.1% Tween-20 (PBST)
• Membrane was placed on shaker for 1 hour and 5% BSA solution was discarded
• Primary antibody (RRID:AB_915950, rabbit polyclonal, Cell Signaling Technology) in 5% BSA solution is added without any washing. Primary antibody is initially used at concentration of 1:1000, unless otherwise stated. Allow membrane to shake overnight at 4 degrees with primary antibody. 
• Primary antibody solution was removed and membrane was washed with PBST for at least 10 minutes per wash for a total of 3 washes. Washing includes adding solution and placing membrane on shaker 
• Secondary antibody is added at a concentration of 1:10,000 in PBST with 5% BSA to membranes. We utilize LICOR IR range secondary antibodies at this concentration. Place membrane on shaker with secondary antibody for 1 hour
  • Secondary should be added based on species the primary antibody was raised in
• Secondary antibody solution was removed and membrane was washed with PBST for at least 10 minutes per wash for a total of 3 washes. Washing includes adding solution and placing membrane on shaker 
• Membrane was read on LICOR scanner using the low sensitivity, western blot setting which produces color image
• Images were exported from LICOR software and analyzed using ImageJ

Materials: 

• CST Cell lysis buffer
• Halt Protease Phosphate Inhibitor
• Pierce BCA protein assay kit
• Invitrogen Bolt Bis-Tris Plus Gel system
• iBlot 2 gel transfer system
• LICOR infrared imaging system

• Please refer to Key Resource Table (in original publication) for more details.