Additional Materials Required
•
Microcentrifuge capable of operating at 1000 × g
•
2mL collection tubes
•
End-over-end mixer
Spin Column Procedure for Top 12 Protein Removal
Note: The concentration of serum/plasma proteins can vary widely depending on the origin of the sample. Each 810µL of antibody resin slurry is optimized to process 10µL (600µg) of serum or plasma. For best results, optimize the ratio of sample to slurry volume for each specific application.
1.
Equilibrate the depletion spin column to room temperature.
2.
Remove the column screw cap and add 10µL of sample directly to the resin slurry in the column.
3.
Cap the column and invert the column several times until the resin is completely suspended in the solution.
4.
Incubate the mixture in the column with gentle end-over-end mixing for 60 minutes at room temperature. Make sure the sample mixes with the resin during the incubation period. Alternatively, vortex every 5 minutes.
5.
Twist off the bottom closure and loosen the cap. Place column into a 2mL collection tube and centrifuge at 1000 × g for
2 minutes.
6.
Discard the column containing the resin.
7.
Filtrate contains sample with the top 12 proteins removed. Use for further processing or store for later use. The depleted sample will be in 10mM PBS, 0.15M NaCl, 0.02% azide, pH 7.4.
8.
Sample processing will depend on the type of downstream analysis and may require buffer exchange and/or concentration for 2D gel electrophoresis and MS analysis. Use Thermo Scientific Pierce Protein Concentrators for buffer exchanging and/or concentration (see Related Thermo Scientific Products Section).
Troubleshooting
Problem Possible Cause Solution
Incomplete removal of albumin or IgG
Sample exceeds binding capacity
Incomplete binding
Sample is not mixed during incubation
Reduce amount of sample processed
Increase incubation time
Mix the sample with resin by gentle end-over-end mixing and make sure that the sample is mixing with the resin during the incubation period.
Related Thermo Scientific Products
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Pierce Top 2 Abundant Protein Depletion Spin Columns, 6 columns
Pierce Top 2 Abundant Protein Depletion Spin Columns, 24 columns
Pierce Normal Human Serum
Pierce Protein Concentrators, 9K MWCO, 7mL
BCA Protein Assay Kit
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General Reference
Anderson, N.L. and Anderson, N.G. (2002). The human plasma proteome: History, character, and diagnostic prospects. Mol Cell Proteomics 1: 845-67.
Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
(815) 968-0747
(815) 968-7316 fax www.thermoscientific.com/pierce
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Pierce Biotechnology
3747 N. Meridian Road
PO Box 117
Rockford, lL 61105 USA
3
(815) 968-0747
(815) 968-7316 fax www.thermoscientific.com/pierce
