Y Genetel Laboratories, LLC

Leader in Proteomics Abs and Abs Arrays1202 Ann Street, Madison, WI 53713 Phone 877-248-4316 Fax 608-257-1385 Email info@genetel-lab.com

GAPAR-Hu Anti-Human Plasma Total Protein Column Kit
Description
GAPAR-Hu is an anti-normal human plasma total proteins column for affinity depletion(removal) of global abundant proteins from the plasma samples to facilitate proteomic
study for identifying new protein markers. The advantage of GAPAR (Global AbundantProteins Affinity Removal) over MARS (Multiple Affinity Removal System) is thatGAPAR removes most, if not all, abundant proteins and retains the low abundant andnon-antigenic proteins while MARS is designed for removing only a few known (up to20) abundant proteins. Depending on the samples, about 90-95% protein mass could beremoved by GAPAR-Hu. GAPAR-Hu is highly recommended for identification of new
plasma protein markers associated with diseases. The column can be re-used for multiple
times after regeneration. The kit is highly suitable for downstream proteomic analysis
methods such as LC-MS/MS. It is easy to use and the whole process will take less thanone hour. The column can be regenerated and re-used many times. We have tested thecolumn and found similar depletion efficiencies after 10 regenerations.
Product Components and Storage Condition
2 GAPAR-Hu spin columns; column size: 0.5ml resins in PBS with 0.05% Na Azide;store at 4oC up to one year; binding capacity is about 2.5mg/ml resin.
3 2mL collecting tubes for spin columns, store at room temperature
1 Microcon Ultracel YM-3 for concentrating proteins, store at room temperature
Material/Equipment needed
Human plasma or serum samples (5ul sample per kit)
PBS with or without 0.05% Na Azide or other suitable buffer that will not interfere
antigen-antibody binding
Elution Buffer: 0.1M Citrate/Na Citrate, pH2.5-3 (15.4g Citric acid, 5.9g Na3 Citrate in
1L DI Water, adjust pH to 2.5)
Microcentrifuge

GAPAR-Hu Protocol
You will need to pass your plasma sample through the two columns to obtain efficient
depletion of abundant proteins. Therefore, you should mark your column (such as 1 and2) in order to remember which one has your sample on. You may do this in cool room or4oC condition if desired.
1. Prepare plasma sample: add 5ul plasma sample into 95ul PBS; filter through a.45μm filter if desired.

Genetel Laboratories, LLC

Leader in Proteomics Abs and Abs Arrays1202 Ann Street, Madison, WI 53713 Phone 877-248-4316 Fax 608-257-1385 Email info@genetel-lab.com

2. Remove the top cap then the bottom cap from the columns to drain the column.
Make sure that there are no resins inside the top cap, and if so, use PBS to rinsethe resins into the columns. Put the bottom cap back after the buffer surface
reaches the top of resins.
3. Apply the 100ul prepared plasma sample onto one column, and 100ul PBS onto
the second column. Put the top cap back.
4. Mix for 20 minutes. You may need to tap the side of column so that resins andsamples are completely mix, and then inverting the columns.
5. After the 20 minutes mixing, loose the top cap with a half turn then remove thebottom cap and put the columns into the 2ml collecting tubes immediately.
6. Spin the columns in a microcentrifuge for 10 seconds.
7. Immediately transfer the flow through plasma sample from the first column into
the second column and mix well by tapping on the side of column. You can add
about 300ul buffer into the first column and mix, which will be used for balancing
the centrifugation later.
8. After 20 minutes of mixing, loose the top cap with a half turn then remove thebottom cap and put the columns into the new 2ml collecting tubes immediately.
Make sure to mark the tube so that you know which one will contain your sample.
9. Spin the columns in a microcentrifuge for 10 seconds.
10. You will obtain about 300ul-400ul column unbound, low abundant proteins with aconcentration of about 50-100ug/ml.
Concentrating low abundant proteinsIf desired, you may concentrate the proteins to your require volume or amount using
the provided Microcon Ultracel YM-3. Transfer your sample into the Microcon, cap
the tube and balance with another 1.5ml tubes for centrifugation. Spin for about 2060 minutes until it reaches your desired amount.
Column Re-generation
Immediately after spinning, add 300-400ul Elution Buffer into the column and mix
well by tapping on the side of column. Spin for 10 seconds and monitor the OD280.
Repeat this at least 5 times until OD280 is less than 0.050. After completing theelution, wash the column 6 times with 300-400ul PBS with 0.05% Na Azide by thesame method. At the last step, reconstitute the column in 300-400ul PBS with 0.05%Na Azide and store the column in 4oC.

Genetel Laboratories, LLC

Leader in Proteomics Abs and Abs Arrays1202 Ann Street, Madison, WI 53713 Phone 877-248-4316 Fax 608-257-1385 Email info@genetel-lab.com

KDa1951429671

AlbuminIg Heavy Chain

4833
Ig Light Chain

2822127.6

MW 1

23SDS-PAGE Gel Analysis of human plasma sample (lane 1), the column-bound proteins(lane 2) and the column flow through proteins (lane 3). Five micro-liter of plasma wasdiluted with PBS to 100ul and loaded on the columns for affinity depletion of abundant
proteins. The total volume of both column-bound proteins and flow-through proteins
were adjusted to 100ul, the same volume as the diluted original plasma sample. Eachlane was loaded with 15ul of respective sample (lane 1: diluted original plasma; lane 2:
eluted column-bound proteins; lane 3: the column flow-through proteins). Arrows
indicate the proteins (weak bands) that did not bind to the GAPAR-Hu column, which
could be low abundant proteins. About 95% of total protein mass was depleted by theGAPAR-Hu kit.