SubscribeThe Easy Way to Remove Protein from your Samples
Several strategies exist for eliminating proteins commonly contained in biological fluids, plasma or serum, that would otherwise interfere with downstream applications and analyses such as:
LC-MS/MS.
A common method is to add a compound that causes the protein to precipitate, usually an organic solvent. Traditionally centrifugation is used to pellet the precipitated protein, so that the supernatant can be recovered for downstream applications. Whilst this method works well it is time consuming and requires much manual manipulation.
The use of protein precipitation as a method for sample preparation is widely used within the pharmaceutical research community for applications such as quantitative drug analysis by LCMS/MS. Consequently the need to automate the technique, to save technician time, has spawned a new generation of special multi-well filtration microplates adaptable for automated sample preparation using liquid handling equipment.
Now from Anachem, the new QPF 96-well Protein Precipitation Filter Plates and Tubes offer ease of-use with unrivalled reliability, whether used manually or as part of an automated system. Simply add acetonitrile to the Anachem QPF tube or well, then add your sample, aspirate, and then apply a vacuum or positive pressure. Internal standard can be added to the plasma or acetonitrile. The filtrate will be free of precipitated proteins. Samples will give a similar ionisation suppression profile to samples centrifuged after protein crash is performed.
Anachem QPF Protein Precipitation Plates and Tubes are:
- Easy to automate - a very simple protocol with minimum liquid handling
- Easy to use manually - 1ml SPE style tubes or 96-well plates
- Reliable - No flow before vacuum or pressure is applied, guaranteed.
Robust filtration design removes high particulate loads without clogging
Robust Construction
The Anachem QPF plates are constructed using an innovative Double Filter System, consisting of a top filter (100-150µm) to remove large particulates and a bottom filter (10µm) to eliminate any small particles. Each filter is specially treated in order to support organic solvents and plasma, without leakage under gravity, allowing protein precipitation to occur.
Reliable Design
This double filter system ensures that large particles are filtered out and do not clog the top filter, while the bottom filter is able to remove additional particulates. Therefore, unlike other 96-well plates, the QPF plate will not clog even with high particulate loads. Each new batch of plates is tested on a Gilson SPE automated system to maintain reliability and eliminate batch-to-batch variation.
Ease-of-Use
The ability to hold both plasma and organic solvents without leakage, enables simple protein precipitation methods to be employed. Filtration after the sample and solvent have been added to the plate, only occurs when vacuum or positive pressure has been applied.
Versatile
QPF plates are suitable for use with manual protocols or with robotic equipment such as the Gilson SPE 215 system (8 samples simultaneously) or the Gilson 925 Workstation (96 samples simultaneously) and perform equally well under positive or negative pressure.
(ATTACHED PICTURES OF PRO PLATE 1, 2 & 3 WILL GO HERE IN ORDER FROM 1-3).
Protein Precipitation - Manual Method
Step 1. Place the QPF protein precipitation plate onto the vacuum manifold containing a collection plate.
Step 2. Add the appropriate volume of acetonitrile to each well, using the suggested ratio below*. The minimum that should be used is 50µl, and the maximum is 1.2ml.
Step 3. Add the plasma (between 15µl & 400µl) or fluid requiring protein precipitation.
Step 4. Incubate as follows:
100µl of plasma - allow the plate to stand for 2 minutes. >100µl of plasma - either aspirate the sample several times or cap the plate and vortex / agitate for 2 minutes.
Step 5. Apply vacuum (15" Hg for 3 minutes) to filter the sample.
*For precipitation of the maximum amount of protein, we recommend a ratio of acetonitrile to plasma of 3:1. However, for sensitivity reasons, less can be used if required.
Automation of Protein Precipitation
Anachem offers many automation options based on Gilson equipment - please call 01582 747501 to discuss your exact requirements.
Manual Liquid Handling for Protein Precipitation
Anchem offers expertise, support, and the widest range of products for the liquid handling steps involved in protein precipitation. For maximum efficiency choose multichannel pipettes, see pages 16-17 to save 30%. For especially viscous samples, the positive displacement MICROMAN pipettes are the best choice. Contact us on 01582 747500 or visit www.anachem.co.uk for further information.
