Aug 18, 2022
Measuring protein concentration using the Merck Millipore Direct Detect Spectrometer
- 1University of Manchester
Protocol Citation: ronan o'cualain 2022. Measuring protein concentration using the Merck Millipore Direct Detect Spectrometer. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv598m6g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 20, 2022
Last Modified: August 18, 2022
Protocol Integer ID: 67153
Keywords: Millipore Direct Detect, Measuring protein concentration, S-Trap buffer, protein estimation, spectrometer, quantitation, Bradford, BCA,
Abstract
This protocol details the procedure of measuring protein concentration using the Millipore Direct Detect spectrometer.
Attachments
iiaebptmp.docx
173KB
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 00:10:00 (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).
Materials
Equipment
Direct Detect®
NAME
Spectrometer
TYPE
Merck Millipore
BRAND
C134681
SKU
LINK
Equipment
Direct Detect® Assay-free Cards
NAME
Merck Millipore
BRAND
DDAC00010-GR
SKU
LINK
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)
Loading samples on card:
Loading samples on card:
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
The Direct detect is located in lab B2075.
There are spotting trays available on the shelf above the Direct Detect (DD).
Note
DD card in spotting tray.
Label the bottom membrane on the card for blank measurement.
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.
Pipette 2 µL of blank into center of the membrane designated as the blank position.
Pipette 2 µL of your pooled sample to be analyzed into center of the three remaining spots.
Note
Take care not to touch the membrane with the pipette tip, as this might tear the membrane.
Surface tension of the sample should be enough to pull it away from the tip onto the membrane.
Software and measurement:
Software and measurement:
5m
5m
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
Data entry
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.
5m
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.
Note
- Previous measurements may be found under the history tab.
- The Direct detect is accurate for the measurement of protein lysates between 0.3 mg/mL and 5 mg/mL . If you obtain a reading that is higher than 5 mg.mL-1, it will not be accurate, because the calibration curve is not linear above this concentration. To obtain an accurate measurement, dilute your sample 1 in 5 and 1 in 10 in SDS S-Trap lysis buffer, and measure again in triplicate.