TITLE: Measuring protein concentration using the Merck Millipore Direct Detect Spectrometer
AUTHORS: ronan.ocualain

[DESCRIPTION]
This protocol details the procedure of measuring protein concentration using the Millipore Direct Detect spectrometer.

[BEFORE_START]

Initial preparation

Before you begin:

Identify the following equipment that you will use:

10uL pipette and pipette tips
Millipore Direct detect sample cards (DDAC00010-GR)
Millipore Direct detect machine (C134681)





[GUIDELINES]
Initial assumptions:
Allow approximately 20 minutes for measuring the protein concentration.
You will prepare a pool of your sample for protein measurement.
You have cell or tissue lysates in S-Trap lysis buffer (5% SDS with 50 millimolar (mM) TEAB pH 7.5). 
Direct detect protein concentration measurement requires at least 6 uL of your sample.
Protein lysates have been reduced and alkylated and clarified by centrifuging at 14000 x g for 10 min (see reduction and alkylation protocol).
You will also need a blank sample to measure against. The blank is the buffer/solvent used to prepare the sample, but there is no protein in it. This should be 5% SDS with 50 millimolar (mM) TEAB pH 7.5 containing 10 millimolar (mM) DTT with 15 millimolar (mM) IAM.
If you wish to do more than a single pooled measurement, you may buy a box of direct detect cards from us using PPMS (https://corefacilities.manchester.ac.uk/?BioMS).
A box costs £82.50 (price correct as of August 2022).


[STEPS]
SECTION: Loading samples on card:
1. To measure the protein concentration of your lysate, place a Direct Detect card on a clean, dry surface (spotting trays for cards are available).
 

 

SECTION: Loading samples on card:
2. Label the bottom membrane on the card for blank measurement.
SECTION: Loading samples on card:
3. Label a clean 0.75 mL Eppendorf tube with “pool”. To it, add 2 µL 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.

SECTION: Loading samples on card:
4. Pipette 2 µL of blank into center of the membrane designated as the blank position.
SECTION: Loading samples on card:
5. Pipette 2 µL of your pooled sample to be analyzed into center of the three remaining spots.
 

SECTION: Software and measurement:
6. 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.


 

SECTION: Software and measurement:
7. Insert the Assay-free card vertically into the instrument card holder with the instrument and card arrows aligned.
 

SECTION: Software and measurement:
8. Click on the measure card button.
SECTION: Software and measurement:
9. The sample concentrations will appear on the screen, along with the statistical analysis and spectrum plot.
SECTION: Software and measurement:
10. 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.