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.