Determination of Protein Concentration
Set up standards
Use BioRad gamma globulin at 1mg/ml (1ug/ul) as the standard. Don’t use the BioRad
BSA if you can help it. This is diluted from 10mg/ml stock (1:10 in MilliQ water: e.g. 100ul
standard + 900ul water)
The BioRad micro assay is done in disposable plastic cuvettes. Reactions are carried out
in 1ml total volume.
Ideally all samples should be done in duplicate, but I strongly recommend that you do the
standards in duplicate.
Std ug/ml
0
2
4
6
8
10
15
20
Vol 1mg/ml
0
2ul
4ul
6ul
8ul
10ul
15ul
20ul
Vol H20
800ul
788ul
786ul
784ul
788ul
780ul
775ul
770ul
Note that careful pipetting will greatly improve your results. If you are concerned about the
pipette you are using, then weigh 10-100ul of H20. One milliliter of water equals one gram. If
you’re off, try using another pipettor. Check to make sure you’re using proper technique.
Add 200ul of the BioRad Protein Assay concentrate (kept in cold box in 617) that has
been filtered through Whatman filter paper. Filter by folding a circular piece of filter paper
(drawer in 616) and placing it in a funnel. The reagent is poured through the filter paper. Collect
filtrate in a graduated cylinder or flask. This can also be stored refrigerated in a closed bottle or
tube.
Immediately after adding the BioRad Protein Concentrate, carefully vortex and make sure
that the reactions are evenly mixed – no brown regions at the bottom of the cuvette.
Incubate at room temperature for at least 5 minutes but not more than 60 minutes
Read absorbance at 595nm on the spectrophotometer or plate reader. Record results by
hand
Enter the readings into Excel and average them. If you’re doing unknown samples you
should include them as well.
Subtract the “0” value from the rest of the standards. Most instruments will do this
automatically by reading the “0” sample as “blank” or “reference”. If you do a manual
background subtraction you must also subtract this value from your unknowns.
Plot the standard conc in ug/ml (X axis) versus the average absorbance readings (Y axis)
using Excel. Add a trendline and get the equation. Important: make sure you plot a “0”reading
which should be “0” for both X and Y axes since the 0ug/ml value was subtracted out. Also get
the r squared value to determine how well the trendline fits the data. Ideally this should be as
close to 1 as possible. R squareds below 0.9 aren’t very good. If you get an r squared below 0.9
you should consider re-doing your standard curve.
Use Excel to solve the equation for “x” and use this equation so solve for your unknowns.
This is done by using the abs readings of the unknowns as “y” and calculating “x” in ug/ml. Be
sure to include any dilutions you made of the unknowns prior to the assay. Note that
concentrations are in ug/ml. I strongly suggest that you convert that into ug/ul. It makes this
much easier. You can check to make sure your calculations are right by plugging in a few of the
“y” values for some of the standards as an unknown and solving for “x”. If you’ve got it right
you should get 10mg/ml (10ug/ul) as the result. This is the concentration of your standard prior
to being diluted 1:10.
Calculate the volume of each unknown that contains 500ug.
Calculate the amount of water needed to add to the 500ug of sample to equal 100ul
Add the water and sample together in 1.5 ml tubes with locking lids. Add 150ul of SDS
sample buffer with beta mercapto ethanol and vortex. The final volume will be 100ul + 150 ul =
250ul total. There is 500ug total protein in 250ul = 500/250 = 2ug/ul. Loading 10ul per lane on
an SDS gel will deliver 20ug total protein per lane. You should be able to load 50 wells with this
much sample = 500ul total /10ul /lane.
Note if you don’t have enough sample to give 500ug, you can scale any of these back. If
you use less sample then you must also scale back the water and SDS sample buffer by the same
proportion.
Boil samples 5-8 minutes in 1.5ml tubes with locking lids (very important since boiling
tends to pop the tubes open). Chill samples on ice for 2 minutes and then spin at top speed in the
microfuge for 2 minutes. Samples are then used immediately or stored at –80 degrees.