Concentrating Protein in Media Samples with Centrifugal Devices

Youssef Farhat, Written 12/28/12, Last updated 5/6/14

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Introduction

NanoSep Centrifugal Device

Concentrating media samples can be necessary when a protein of interest is present at concentrations below the detectable limit of a given assay.  One quick and convenient way to do this is with the use of centrifugal devices.  The centrifugal devices used here are available from Pall Corporation and come with a variety of different molecular weight cutoffs (MWCO).

We used these devices to concentrate 250 µL of samples containing MMP-2 (50-70 kDA) a total of ten-fold (final volume of 25 µL) for use in gelatin zymography.  We selected a MWCO of 10 kDA, as experience showed that 30 kDa was not a small enough cutoff to prevent MMP-2 from passing through the membrane.

As a word of caution, centrifuging samples for too long can result in lower molecular weight proteins being lost (even if they are above the MWCO), as they can embed themselves in or even pass through the filter-membrane.  It is therefore best to first try some practice samples in order to optimize the centrifugation time such that you don’t centrifuge so little that the media gets insufficiently concentrated, or too much such that too much of your protein of interest gets lost.

Materials

• Nanosep® Centrifugal Devices with Omega™ Membrane, 10K MWCO (Pall Corporation, #OD010C34)
• Molecular grade water (Gibco, #10977), or any suitable solution for resuspending concentrated samples
• Typical lab equipment, such as a microcentrifuge, micropipettors, pipette tips, Eppendorf tubes, and cDNA tubes

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Procedure

1. Media samples should be thawed and kept on ice.
2. Transfer 250 µL of media to the top chamber of a Nanosep centrifugal device.
3. Centrifuge the sample at 14,000 g for 10 minutes at 4°C.
4. Check the top reservoir to ensure that the majority of sample solvent has passed through the membrane.
   1. If a significant droplet remains above the membrane, centrifuge at 14,000 g for another 5 minutes and check again.  Repeat this step up to three additional times if necessary (for a maximum of 30 minutes of centrifugation).
5. Add 25 µL of molecular grade water to the top of the reservoir and pipette up and down several times around the surface of the reservoir membrane.  Avoid making bubbles as much as possible.
6. Transfer 25 µL to a new Eppendorf tube and perform desired assay with 10X concentrated sample.
   1. If 25 µL is difficult to recover, only 20-24 µL may be recoverable (this often occurs when many bubbles are produced during pipetting.  Do your best to avoid making bubbles!)

Citing this Protocol

Farhat, Y. “Concentrating Protein in Media Samples with Centrifugal Devices.” The Protocol Place. Dec 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com/>