Measuring protein concentration using the Merck Millipore Direct Detect Spectrometer

To measure the protein concentration of your lysate, place a Direct Detect card on a clean, dry surface (spotting trays for cards are available).

Note

Label the bottom membrane on the card for blank measurement.

Label a clean 0.75mL Eppendorf tube with “pool”. To it, add 2µL of each of your samples. When complete, vortex mix briefly. A pooled sample should be representative of the batch you wish to measure, such as a set of replicates.

Pipette 2µL of blank into center of the membrane designated as the blank position.

Pipette 2µL of your pooled sample to be analyzed into center of the three remaining spots.

In the software, complete the fields as follows:

a. User name should be preset (Facility).

b. Card name – today’s date, followed by your initials and the sample number.

c. Protein – use the drop down menu to select “proteins in S-Trap buffer.q3”.

d. Ensure that the box marked “ dry sample card ” is ticked.

e. Do not tick the boxes marked “lipid”, “extended drying cycle”, “use previous blank”, or “modified mass unit”.

f. Give the 4 card positions a name, the position marked 1 in blue is the blank , while the other three should be in green, these are your three replicate measurements.

Note

Insert the Assay-free card vertically into the instrument card holder with the instrument and card arrows aligned.

Note

Make sure that the "M" writing side is facing the left. The instrument will move the card up and down and sound a tone. The green light illuminates to confirm proper insertion.

Click on the measure card button.

The sample concentrations will appear on the screen, along with the statistical analysis and spectrum plot.

After all four positions on the card have been read, the instrument will sound a tone. The card will rise to the initial insertion position. Remove the card and dispose of it.