Thermo Scientific Zeba, 89878, 89882, 89883, 89877 Instructions Manual

INSTRUCTIONS

Pierce Biotechnology

PO Box 117

[phone]

thermofisher.com

3747 N. Meridian Road

Rockford, lL 61105 USA

[phone] fax

Pub. No. MAN0015751

Pub. Part No. 2162579.1

Rev A.0

Zeba Spin Desalting C olumns and Plates,
7K MWCO

Number Description

89877 Zeba Spin Desalting Columns, Micro (75µL), 25 columns, for 2-12µL samples
89878 Zeba Spin Desalting Columns, Micro (75µL), 50 columns, for 2-12µL samples
89882 Zeba Spin Desal ting Columns, 0.5mL, 25 columns, for 30-130µL samples
89883 Zeba Spin Desalting Columns, 0.5mL, 50 columns, for 30-130µL samples
89889 Zeba Spin Desalting Columns, 2mL, 5 columns, for 200-700µL samples
89890 Zeba Spin Desalting Columns, 2mL, 25 columns, for 200-700µL samples
89891 Zeba Spin Desalting Columns, 5mL, 5 columns, for 500-2000µL samples
89892 Zeba Spin Desalting Columns, 5mL, 25 columns, for 500-2000µL samples
89893 Zeba Spin Desalting Columns, 10mL, 5 columns, for 700-4000µL samples
89894 Zeba Spin Desalting Columns, 10mL, 25 columns,
89807 Zeba Spin Desalting Plates, 96-well, 2 plates
89808 Zeba Spin Desalting Plates, 96-well, 4 plates

Note: These products are recommended for processing compounds > 7000Da. The resin slurry is

Storage: Upon receipt store at 4°C. Product shipped at ambient temperature.

Introduction

The Thermo Scientific™ Zeba™ Spin Desalting Columns contain a high-performance resin that offers exceptional desalting
or buffer-exchange for protei n s amples. Samples volumes between 2µL-4mL and containing as low as 20µg of protein/mL
can be processed with unsurpassed protein recovery and ≥ 95% retention of salts and other small molecules (< 1000Da).
These columns require no chromatography system. The s pin-column method eli mina te s waiting for samples to emerge by
gravity flow and the subsequent monitoring o f fractions for prot ein recovery.

Important Product Information

• The Zeba Spin Desalting Columns and Plates contain a size-exclusion chromatographic resin to separate proteins from

smal l molecules. As wit h all size exclusion-based separation, the amount of small molecule removed and protein
recovered are affected by the nature of the molecules and volume of sample. The sample volumes recommended provide
exceptional removal of a variety of small molecules (typically > 95% for molecules < 1000Da); however, proteins and
small molecules often behave differently than predicted because of a variety of factors such as hydrophobicity,
secondary structure and interactions. Therefore, some opti mization of sample volume might be required to achieve
optimal performance for each specific sample. In general, reducing the sample volume added to the column increases
small molecule removal, and increasing sample volume maximizes protein recovery.

for 700-4000µL samples

supplied in 0.05% sodium azide.

• Also available are Zeba Spin Desalting Columns and Plates with a 40K MWCO, which ena ble removal of salts and other

small molecules < 2000Da and recovery of proteins and other macromolecules > 40,000Da.

Pierce Biotechnology

PO Box 117

[phone]

thermofisher.com

3747 N. Meridian Road

Rockford, lL 61105 USA

[phone] fax

2

Table 1. Centrifugation times and volumes for the buffer, stacker and sample.

Column or Plate

75µL

0.5mL

2mL

5mL

10mL

Plate

Sample Volume Range (µL)

Wash/equilibration Buffer Volume

50µL

300µL

1mL

2.5mL

5mL

250µL

Sampl e Volume (µL)*

< 5

< 70

< 350

< 750

< 1500

< 30

Optional Stacker Volume (µL)*

Centrifuge Speed (× g)

Storage Sol ution

Removal

Wash 1

1 1 2 2 2

2

Wash 2

1 1 2 2 2

2

Wash 3

1 1 2 2 2

2

Sample Recovery

2 2 2 2 2

2

*When using the indicated sample volumes, use a stacker to achieve the highest recovery. The stacker is a volume
of wash/equilibration buffer applied after the added sample has completely entered the desalting resin bed.

Procedure for Desalting or Buffer Exchange

Additional Materials Required

• For 75µL and 0.5mL spin columns: Bench-top microcentrifuge (1500 × g) and 1.5mL microcentrifuge tubes

Note: Use a centrifuge that can be adjusted to 1500 × g, such as the Thermo Scientific™ Sorvall™ Legend Micro 17
Microcentrifuge

• For 2 and 5mL spin columns: Centrifuge (1000 × g) and 15mL conical tubes

• For 10mL spin columns: Centrifuge (1000 × g) and 50mL conical tubes

• For the desalting plates: Varia ble-speed centrifuge with rotor and carrier capable of handling stacked plates

(height = 4.4cm) at 1000 × g

• Wash/equilibration buffer

Note: Use the same wash/equilibration (stacker) buffer as is desired for the final sample solution. Equilibrating the
desalting resin before sample loading is necessary to ensure proper buffer exchange.

Procedure for Protein Desalting

Note: See Table 1 for centrifugat ion times and volumes for the buffer, stacker and sample for each device. For maximum
protein recovery, add a stacker on top of the applied sample for volumes below the volume specified in Table 1.

1. Remove the column’s bottom closure or the plate’s bottom sealing material. Loosen cap (do not remove cap).

2. Place the column into a collection tube or plate on top of a wash plate and centrifuge to remove the storage solution.

3. Discard flow-through and replace the column back into the collection device.

4. Add wash /equilibration buffer on top of the resin. Centrifuge device and discard flow-through. Repeat this step two

additional times.
Note: After each spin, the resin should appear white and free of liquid. If liquid is present, make sure you are using the

correct centrifugation speed and time. Incomplete centrifugation may result in poor sample recovery or sample dilution.

5. Blot the bottom of the column or plate to remove excess liquid. Transfer device to a new collection tube or plate.

6. Apply sample on top of the resin. If needed, add a stacker as soon as the sample has entered the resin. Adding a stacker is

optional but recommended for dilute pr otein solutions or small sample volumes to ensure maximum sample recovery.

7. Centrifuge and retain flow-throug h that contains sample. Discard spin column or plate.

2-12 30-130 200-700 500-2000 700-4000 20-100

3 15 40 100 200 10

1000 1500 1000 1000 1000 1000

1 1 2 2 2 2

Centrifugation

Time (min)

Pierce Biotechnology

PO Box 117

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thermofisher.com

3747 N. Meridian Road

Rockford, lL 61105 USA

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Problem

Possible Cause

Solution

Sample or buffer does not

Centrifugation problem

Ensure that c entrifuge is in prop er working co ndition

Ensure bottom closure i s removed

Ensure top cap is loosened

Sample contamination

Improper sample loading
Apply sample directly to center of the resin bed; touch tip

Avoid contact with sides of column

Improper centrifugation
For fixed-angle rotors, place colu mn in the same orientation

Do not exceed recommended centrifugation speed or time

Low yield

Sample was not completely in
Portion of protein still

Use a stacker to recover more protein (for most samples,

Protein precipitated in

Check for protein solubility in the final b uffer or solution

Protein bound to resin matrix

Use alternative resin, such as Polyacrylamide Spin

Recovered protein or

Wash/equilibration buffer was

Before adding the sample make sure the wash/equilibration

Troubleshooting

flow through resin

solution before adding to the
column

to resin to expel all sample

each time and do not exceed recommended centrifuge speed

Centrifuge sample at 14,000 × g for 10 minutes before
adding to the column

remaining in spin column

the majority of protein is recovered without a stacker)

equilibration buffer

Desalting Colu mns (see Related Thermo Scientific Products
section)

sample is dilute

not adequately removed

buffer was adequately removed by centrifugation (i.e.,
column appears uniformly white with no solvent streaks)

Related Thermo Scientific Products

89849 Pierce Polyacrylamide Spin Desalting Columns, 7K MWCO, 0.7mL, 25 columns
89862 Pierce Polyacrylamide Spin Desalting Columns, 7K MWCO, 0.7mL, 50 columns

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warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to
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