FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

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1 User Protocol Rev. 12 May 2005 JSW Page 1 of 7 ProteoExtract Albumin/IgG Removal Kit, Maxi Cat. No Introduction One of the major challenges in functional proteomics is the handling of complex samples prior to comparative analysis e.g. for disease marker identification. The best sources for potential disease marker identification are body fluids because they are easily available in sufficient amounts for analysis. However, the main drawback of a comprehensive analysis of body fluids is the high abundance of serum albumin and immunoglobulins. Serum albumin can constitute 50-70% of the total serum protein and immunoglobulins constitutes 10-25%. The high concentration of these proteins causes loss of resolution during electrophoresis and other chromatographic separations. The ProteoExtract Albumin/IgG Removal Kit, Maxi provides a convenient, reproducible and highly specific method to remove serum albumin and immunoglobulins from body fluid samples. The kit provides columns containing either a novel albumin-specific affinity resin or a proprietary immobilized protein A polymeric resin. These columns allow for the removal of more than 80 % of both serum albumin and immunoglobulins from human body fluid samples when used according to the instructions provided in this manual. The column hardware is designed to allow for both manual and LC-instrument controlled usage. When used manually, the columns are operated as syringe-tip filters, when used with LC-instrument control, the columns can be connected to any standard HPLC/FPLC instrument via a 10/32 port using the provided adapters. For guidelines of column connections and operation, please refer to section 3 of this manual. The affinity resins in the kit have been developed for highly specific binding to either serum albumin or immunoglobulins and show very low background binding of other serum or plasma proteins as demonstrated by LC-MS analysis of captured proteins (not shown) and immunoblotting against marker proteins (see Figures 1A and 1B). The marker proteins Transferrin and Antithrombin III could be recovered nearly quantitatively in the unbound fraction of the respective column. Concurrently, in a typical experiment using the two types of removal columns consecutively to deplete human serum from both albumin and immunoglobulins, more than 80 % of high abundance proteins were removed from human plasma as monitored by densitometry of stained bands after 1D- PAGE separation of respective samples (Fig.1C). 1 A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt,

2 User Protocol Rev. 12 May 2005 JSW Page 2 of 7 Fig. 1A P U U B B Fig. 1C M P U Blue A M Fig. 1B 50 kda Albumin IgG Heavy Chain Fig 1: The ProteoExtract Albumin/IgG Removal Kit (Maxi) contains columns filled with affinity resins especially developed for binding of either albumin or immunoglobulin with very low background binding to other plasma proteins. To demonstrate resin specificity, human plasma was subjected to albumin removal (Fig.1A) or IgG removal (Fig.1B). Samples were processed as described in the kit procedure. 5 µg protein from human plasma (P), the respective unbound (U) or bound (B) fractions were separated by 1D-SDS-PAGE and visualized by probing with anti-transferrin antibody (upper panel) and anti-antithrombin III antibody (lower panel). More than 95 % of both marker proteins were recovered both with Albumin - and IgG removal columns. Fig. 1C: Using this Removal Kit, more than 80 % of both albumin and immunoglobulins can be simultaneously removed from plasma and serum samples. 15 µg protein from human plasma (P) and the albumin/igg-depleted unbound plasma fraction (U) as well as the eluate fractions from albumin removal column (Blue) and Protein A columns (A) were separated by 1D-SDS-PAGE and visualized by Coomassie staining. The albumin removal column contains a resin that has been optimized for the removal of albumin from human samples. However, the provided columns will also bind albumins from other species although with lower efficiency. The dynamic binding capacity of one column was determined to be 6.1 ± 0.3 mg human albumin in Binding Buffer at a flow rate of 0.1 ml/min and 10 % breakthrough. To provide guidelines for different species we have evaluated the relative amount of albumin bound to the albumin removal column (Table 1). Albumin Species Bound albumin [% of total] Ratio to Human Albumin Reference Sample Human 86 - Rabbit 86 1 Rat Mouse Pig Bovine The Binding Buffer provided with the kit has been optimized to allow for specific binding of both albumin and immunoglobulins to the respective column without requirement of buffer exchange. This allows for the subsequent use of the two types of columns either independently using the flow through of the albumin removal column as sample load for the IgG removal column or with the two types of columns being directly connected in a row (see 3.1 and 3.2). 2 A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt,

3 User Protocol Rev. 12 May 2005 JSW Page 3 of 7 The IgG Removal Column contains Protein A which is a highly stable surface receptor produced by Staphylococcus aureus. Protein A binds the Fc portion of IgGs from a large number of species 1. The dynamic binding capacity of the Protein A column was determined to be 4.5 mg ± 0.2 mg human IgG in Binding Buffer at a flow rate of 0.1 ml/min and 10 % breakthrough. The provided removal columns are guaranteed to be functional as indicated when bound proteins are eluted with the provided removal buffers and columns are used according to the manufacturers instructions given in this manual. Kit Components (for 20 samples of µl body fluid) 1) Albumin Removal Column, Maxi (Cat. No. KP31640) 2 items with Luer lock adapters 2) IgG Removal Column, Maxi (Cat. No. KP31650) 1 item with Luer Lock adapter 3) Albumin/IgG Binding Buffer 10X (Cat. No. KP31641) 1 vial: 90 ml 10 x buffer 250 mm sodium phosphate, ph 7.4 4) Albumin Removal Buffer (Cat. No. KP31642) 1 vial: 70 ml buffer 25 mm sodium phosphate, ph 8.0; 2 M NaCl 5) IgG Removal Buffer (Cat. No. KP31651) 1 vial: 70 ml buffer 250 mm citric acid 2. Storage Conditions For long term storage the kit should be stored at 4 C. Avoid freezing of the ProteoExtract Blue and ProteoExtract Protein A columns! Freezing and thawing will destroy the affinity resin and the columns will no longer function. When stored at 4 C all kit components are stable for at least 12 months. 1 Boyle, M. D. P. and K. J. Reis., Biotechnology 1987, 5, A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt,

4 User Protocol Rev. 12 May 2005 JSW Page 4 of 7 3. Technical Hints and Preparation Instruction 3.1 Operation of removal columns One removal column has a column volume (CV) of 1 ml. Columns can be used under both manual and LCinstrument control. When used manually, the columns are operated as syringe-tip filters. When used under LCinstrument control, connect the column to a 10/32 port on a standard HPLC/FPLC instrument. Please refer to the drawing below for instructions of set-up. All connections of the column and adapters must be finger-tight. Applying too much force when closing the connections will impair with the column function and may cause leakage. Manual Usage: LC-instrument Usage: Remove the black plug from the upper end of the column. Connect the provided Luer- Lock adapter by screwing into the stopper. Remove transparent lower plug. The column is ready for manual operation as syringe tip filter. Two albumin removal columns may be connected to raise the column capacity if desired. Remove the transparent lower plug from the lower end of the column. Connect the provided Luer-Lock adapter by screwing it into the Luer-Lock fitting at the end of the column. The column/adapter setup can now be inserted into a 10/32 port on a LCinstrument valve. Several columns may be connected to raise the column capacity if desired. 4 A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt,

5 User Protocol Rev. 12 May 2005 JSW Page 5 of In order to deplete body fluid samples from both albumin and immunoglobulins, link the IgG Removal Column downstream to the Albumin Removal Column(s) using the provided Luer-lock adapter. 3.3 For sample dilution, use the 10X Binding Buffer without prior dilution according to the examples boxed below. 90 µl body fluid + 90 µl 10X Binding Buffer µl high quality water = 900 µl diluted sample 180 µl body fluid µl 10X Binding Buffer µl high quality water = 1800 µl diluted sample 3.4 To prepare a 1X Binding Buffer dilute an appropriate volume of the 10X Binding Buffer 10-fold with high quality water (1 volume 10x buffer + 9 volumes water). 4. Reagents and Equipment not provided Micropipettes and tips, 10 µl, 200 µl and 1 ml size Syringes with different volumes for manual usage. High quality water (resistance > 18 mω) Sample collection tubes e.g. 15ml centrifuge tubes 5. Protocol Guidelines Wear gloves, safety glasses and lab coats during the procedure. Follow your facilities safety standards for handling potentially biohazardous samples. The procedure for albumin and IgG removal from body fluids takes approximately 20 min by manual operation. The time required for LC-instrument controlled runs depend on the individual program used. The washing steps of the protocol are required to increase the yield. However, if sample concentration is critical, the washing steps may be omitted but then non-specific binding will become more evident. One Albumin Removal Column has a capacity of at least µl of plasma or serum One IgG Removal Column has a maximum capacity of 400 µl of plasma or serum. Thus, two runs of albumin removal can be loaded onto one IgG removal column. Note: For elution of bound proteins both types of columns require different elution buffers. Column assemblies have to be disconnected. Use of the wrong elution buffer may cause damage to the resin and impair the column function. Columns are working optimal with flow rates between 0.1 ml/min and 0.5 ml/min. As a good starting point a flow rate of 0.25 ml is recommended. During LC instrument controlled runs do not exceed maximum flow rate of 1 ml/min. All reagents and manipulations can be performed at 4 C or at room temperature. 5.1 Protocol 1: Removal of albumin and immunoglobulins from body fluids (manual operation) 1. Dilute the desired amount of sample with 10X Binding Buffer and water as indicated in section A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt,

6 User Protocol Rev. 12 May 2005 JSW Page 6 of 7 2. Mount a column or a column assembly for syringe tip filter usage as indicated in section Fill a syringe with ml of 1X Binding Buffer for column equilibration. Remove any trapped air from the syringe before connecting with the column. 4. By application of gentle pressure allow a volume of 2 ml 1X Binding Buffer per column to pass through the resin bed. Discard the flow through. 5. Fill a new syringe with the diluted sample. By application of gentle pressure allow diluted sample to pass through the column. Collect the column(s) flow-through. Note: Count to five in between two consecutive droplets to allow for sufficient contact time between the sample and the resin. 6. Connect the syringe of step 3 filled with 1X Binding Buffer to the column(s). By application of gentle pressure allow a volume of 2 ml buffer per column to pass the resin bed. Collect the column(s) flow-through as the wash fraction. Combine wash fraction with the previously collected flow-through as Depleted sample. 7. Unmount column assemblies prior to elution of bound proteins from albumin- and IgG removal columns. Fill one syringe with Albumin Elution Buffer, another with IgG Elution Buffer and connect the syringes to the apropriate columns. 8. Allow 2 ml of the individual Elution Buffer per column to pass the resin bed. Optional: Collect the eluates 9. Re-equilibrate the individual columns with 2 ml 1X Binding Buffer as indicated in step 4 and store at 4 C. 10. For preparation of depleted samples in downstream analysis please refer to the Technical Appendix in section Protocol 2: Removal of albumin and immunoglobulins from body fluids (LC-instrument controlled operation) 1. Dilute the desired amount of sample with 10X Binding Buffer and water as indicated in section Mount a removal column or a column assembly for LC-instrument control usage as indicated in section Prepare sufficient amounts of 1X Binding Buffer, Albumin Removal Buffer and IgG Removal Buffer. In case the provided buffer volumes are not sufficient for your experiments, please refer to section "Kit components" for recipes. 4. Equilibrate your LC system with the Buffers as indicated in the respective instruction manual. 5. Columns are operated at flow rates between 0.1 ml/min and 0.25 ml/min for abundant protein removal. A flow rate of 0.25 ml is generally recommended when using µl sample per column and when using column assemblies. With higher sample loads per column or higher concentrations of albumin in the sample, lowering the flow rate to 0.1 ml/min is recommended. 6. Use the following guidelines for programming your LC instrument for albumin and IgG depletion from body fluids: a. Equilibrate column or column assembly with 3 CV's. b. Inject diluted sample; collect flow through c. Wash out unbound sample with 4 CV s of 1X Binding Buffer. Collect wash fraction. d. Elute each type of column with 2 CV s of the appropriate Removal Buffer (unmount column assemblies prior to elution). Optional: Collect eluates. 6 A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt,

7 User Protocol Rev. 12 May 2005 JSW Page 7 of 7 e. Reequilibrate columns immediately after elution with 3 CV s of 1X Binding Buffer. f. Combine flow through and wash fractions as Depleted sample 7. For preparation of collected fractions for downstream analysis please refer to (6) Technical Appendix 6. Technical Appendix Preparing the depleted sample for further analysis Protein Quantification Absorbance at 280 nm can be used to estimate the protein content. For more accurate protein quantification, any method not influenced by the presence of acetate buffer can be used; e.g. BCA Protein Assay Kit (Cat.No.71285) or the Non-Interfering Protein Assay Kit (Cat.No ). One-dimensional Electrophoresis Mix sample with an equal volume of 2x gel loading buffer (not provided: e.g. 125 mm Tris/HCl, ph 6.8; 10 % (w/v) SDS; 30 % (v/v) Glycerol; 100 mm DTT; 0.002% (w/v) bromophenol blue) and heat samples at C for 5 min prior to loading on SDS-PAGE. Please refer to the gel equipment manufacturers instructions for details. One-dimensional gel electrophoresis can usually be performed without pre-concentration of the albumin and IgG-depleted sample. Two dimensional Gel electrophoresis Two-dimensional electrophoresis of albumin and IgG-depleted serum/plasma yields optimal results after removing excess salts following precipitation of proteins with e.g. TCA/Acetone (Yuan et al., 2002: Electrophoresis 23: ): Adjust albumin and IgG-depleted sample to a final concentration of 20% TCA and 80% Acetone by adding four volumes of a solution of 25% TCA in acetone. Incubate at 4 C for 1h. Spin briefly and discard supernatant Wash the pellet twice with 100 µl acetone. Air-dry pellet and re-dissolve directly in suitable IEF buffer. Please refer to the two dimensional gel equipment manufacturers instructions for details. Liquid chromatography Albumin/IgG-depleted plasma/serum samples can be applied directly to any liquid chromatography technique not influenced by the presence of low concentration of phosphate buffer. If buffer exchange or concentration is required, subject samples to ultra filtration. 7 A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt,