C18 Spin Column Usage Method

597PR-01
G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected]
A Geno Technology, Inc. (USA) brand name
C18 Spin Columns

For Purification and Concentration
of Peptide Samples
(Cat. # 786-930, 786-931)
think proteins! think G-Biosciences www.GBiosciences.com

INTRODUCTION ................................................................................................................. 3
ITEM(S) SUPPLIED .............................................................................................................. 3
STORAGE CONDITIONS ...................................................................................................... 3
SPECIFICATIONS ................................................................................................................. 3
ADDITIONAL COMPONENTS REQUIRED............................................................................. 3
IMPORTANT INFORMATION .............................................................................................. 4
PREPARATION BEFORE USE ............................................................................................... 4
PROTOCOL ......................................................................................................................... 5
A. SAMPLE PREPARATION.............................................................................................. 5
B. PREPARE C18 SPIN COLUMNS ................................................................................... 5
C. PEPTIDE BINDING ...................................................................................................... 5
D. WASH & PEPTIDE RECOVERY ..................................................................................... 5
TROUBLESHOOTING .......................................................................................................... 6
RELATED PRODUCTS .......................................................................................................... 7
Page 2 of 8
INTRODUCTION
G-Biosciences C18 Spin Columns are ready-to-use micro centrifuge columns for peptide
clean up and concentration. The columns consist of porous C18 reverse-phase resin
that has a particle size if ~15µm and a pore size of 300Å. The resin offers highly efficient
binding and recovery of peptides and is ideal for mass spectrometry and other peptide
related applications.
Each spin column can be used to process between 10 to 150µl peptide samples in about
30 minutes without the need for specialized equipment. Each column can bind between
10ng to 30µg of protein peptides, although sensitivity and detection limits are
dependent on selected downstream applications.
ITEM(S) SUPPLIED
Cat. # Description Size
786-930 C18 Spin Columns 25 columns
786-931 C18 Spin Columns 50 columns
STORAGE CONDITIONS
Shipped at ambient temperature. Upon receipt store at room temperature.
SPECIFICATIONS
• Binding Capacity: 10ng-30µg protein peptides
• Volume Capacity: 10-150µl
• Support:C18 coated silica gel
• Particle size: 15µm
• Pore size: 300Å
ADDITIONAL COMPONENTS REQUIRED

• Ultrapure water (G-Biosciences Proteomic Grade Water, Cat. # 786-229)
• Acetonitrile (ACN)
• Tirfluoroacetic acid (TFA)
• 1.5ml microcentrifuge tubes for collection tubes
• Methanol
• Bench top microcentrifuge (Up to 3,000xg)
Page 3 of 8
IMPORTANT INFORMATION
• The lower level of detection for a protein is 20ng (300fmol). Each singular peptide,
at this lower level of detection, needs to be at least 0.5ng to be detected
effectively.
NOTE: Sensitivity and detection limits are dependent on selected downstream
applications
• Free, excess organic solvents (acetonitrile (ACN) or methanol) must be removed for
optimal binding to G-Biosciences C18 spin columns. Simply air dry the sample in a
vacuum evaporator and then carefully resuspend in 20µl 0.5% TFA in 5% ACN.
• Avoid excessive drying of the resin between steps.
• Plastics, including collection tubes and pipette tips, used in the procedure may
introduce contaminants that interfere with mass spec analysis and other
downstream applications. Use high quality plastics (Proteomic Grade Tubes, Cat. #
786-300). Alternatively, treating with Protein-OUT™ (Cat. # 786-680), a unique
solution to remove proteins and other mass spectrometry interfering agents
PREPARATION BEFORE USE

• Activation Solution: 50% Methanol, 400µl/sample
• Equilibration Solution: 0.5% TFA in 5% ACN, 400µl/sample
• Sample Buffer: 2% TFA in 20% ACN, 1µl for every 3µl sample
• Wash Solution: 0.5% TFA in 5% ACN, 400-800µl/sample.
NOTE: Wash volume is dependent upon amount and type of contaminants present
in the sample
• Elution Buffer: 70% ACN, 40µl/sample
NOTE: Acceptable elution buffers include 50-70% ACN with or without 0.1% TFA, 50-
70% methanol with or without 0.1% TFA. 0.1% formic acid can replace the TFA in
ESI-MS applications.
Page 4 of 8
PROTOCOL
A. Sample Preparation
1. Mix 3 parts sample with 1 part Sample Buffer to give a final concentration of 0.5%
TFA in 5% ACN.
NOTE: Each column can only process 10-150µl diluted sample.
B. Prepare C18 Spin Columns

1. Tap the column to ensure the resin is settled in the base of the column, and then
remove the top and bottom caps. Place column in clean collection tube.
2. Add 200µl Activation Solution (50% Methanol) to rinse the walls of the column and
wet the resin.
3. Briefly centrifuge (1,500xg for 1 min) and discard the Activation Solution. Repeat
the Activation Solution wash once.
4. Add 200µl Equilibration Solution (0.5% TFA in 5% ACN), centrifuge as before and
discard solution. Repeat Equilibration Solution wash once.
C. Peptide Binding
1. Transfer the column to a clean collection tube and apply the sample from section
A1 to the top of the resin bed.
2. Centrifuge at 1,500xg for 1 minute and recover the flow-through.
3. Reapply the flow-through to the resin bed to ensure complete binding. Repeat the
centrifugation.
NOTE: Retain the final flow-through to confirm sample binding.
D. Wash & Peptide Recovery

1. Transfer the column to a clean collection tube and apply 200µl Wash Solution
(0.5% TFA in 5% ACN) to the resin.
2. Centrifuge at 1,500xg for 1 minutes and discard the flow-through. Repeat the wash
step once.
NOTE: If sample contains high levels of contaminants then repeat wash step 1-2
times more. Contaminants, such as 2M urea and >100mM ammonium bicarbonate,
will require these additional washes.
3. Transfer the column to a clean collection tube. See Important Information section
about plastics.
4. Add 20µl Elution Buffer (70% ACN) to the resin bed and centrifuge at 1,500xg for 1
minute. Repeat the elution step once.
5. Dry the sample in a vacuum evaporator and then proceed with your established
methods.
Page 5 of 8
TROUBLESHOOTING
Issue Possible Cause Solution
High pH; lack of ion-
Ensure TFA was added (Section A1)
pairing agents
Excess organic Dry sample and resuspend in 20µl 0.1-0.5%
solvent was present TFA
Incomplete or Sample lacks
poor peptide hydrophobicity to None
binding bind C18 resin
Ensure resin does not dry out during the
Resin dried out
procedure. If necessary, keep resin in
before sample
Equilibration Solution until sample ready to
addition
add.
Highly hydrophobic
Use 70% ACN with 0.1% TFA to elute
sample
Binding to plastics can cause significant loss
Incomplete or Sample loss due to
at very low peptide concentrations.
poor peptide non-specific
Minimize contact with plastics and storing
recovery interactions
at<300fmol concentration.
Detection limits of Ensure sample is within the detection limit
application of downstream application.
Page 6 of 8
RELATED PRODUCTS
Download our Sample Preparation and Mass Spectrometry Sample Prep Handbooks.
http://info2.gbiosciences.com/complete-sample-preparation-handbook
http://info2.gbiosciences.com/complete-mass-spectrometry-sample-preparation-
handbook
For other related products, visit our website at www.GBiosciences.com or contact us.
Last saved: 5/19/2015 CMH
Page 7 of 8
www.GBiosciences.com
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597PR-01

G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected]

A Geno Technology, Inc. (USA) brand name

C18 Spin Columns

(Cat. # 786-930, 786-931)

think proteins! think G-Biosciences www.GBiosciences.com

786-930 C18 Spin Columns 25 columns

786-931 C18 Spin Columns 50 columns

ADDITIONAL COMPONENTS REQUIRED

PREPARATION BEFORE USE

B. Prepare C18 Spin Columns

D. Wash & Peptide Recovery

Last saved: 5/19/2015 CMH

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