Protein Assay
Thaw cells from the –80°C fridge
Remove the PBS from on top of the pellet
Add 50ul of the Lysis buffer (Net-N Buffer)
Vortex and Break up pellet
Place on ice for 5 min, again make sure that it is well broken up
Spin down at 4°C at 7000rpm (which is almost max speed for that centrifuge) for 15 min
-Protein assay preparation
NEED SIX TUBES HERE TO GENERATE A STANDARD CURVE
Lable tubes 0, 5, 10, 15, 20, & 30
Add to each of these tubes either 0, 5, 10, 15, 20, 30ul of BSA 1ug /ul
BSA IS ON THE BENCH OF STOCK IS IN THE FREEZER
Add 20ul of 0.5N KOH to these tubes  you hydrolize the BSA with this
-Take samples off of the centrifuge and take off the supernatant from these tubes and place
them on new labled tubes
PLACE THESE TUBES ON ICE
Place 5ul of the supernatant from each tube into 20ul of 0.5N KOH
ADD 1ml OF PROTEIN ASSAY SOLUTION TO ALL OF THE TUBES
You can find this solution on the shelf
Use the 0ul BSA tube to zero the machine and read values in lambda
Record these values to generate your standard curve
Use this standard curve to figure our the concentration of your samples based on their lambda
value
AFTER PROTEIN ASSAY
Add a 33% volume of 4X LAEMLI BUFFER (HOT)
Heat to 85°C for 2 min
Samples are ready for loading
If looking at expression levels MAKE SURE YOU LOAD THE SAME ug OF PROTEIN IN
EACH WELL