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PTMScan® Peptide Purification Kit #35741

No. Size Ordering Information
35741S 1 Kit (10 Purifications) In Stock ptmscan@cellsignal.com
Products Included Quantity Cap Color

PTMScan® Services

View sample data from a PTMScan® study using the Motif Antibody in this kit, provided in a PTMScan® Service report.

Directions For Use

Notes:

• Approximately 20 mg of protease digested peptides can be purified on one C18 reversed-phase column.

• The steps described in the Directions fo Use section use gravity feed. In cases where the flow rate has dramatically decreased, you may apply gentle pressure to the column using the syringe plunger.

• Each time a solution is applied to the column, an air bubble can form at the narrow junction between the syringe and the column inlet. This will decrease the flow rate considerably. The bubble should be removed by flushing with a gel loading tip fitted on a P200 pipette.

1. Acidify the peptides prior to loading by adding 1/20 volume of 20% TFA for a final concentration of 1% TFA.

2. Clarify the peptide sample by centrifuging at 1,780 x g at room temperature for 15 minutes.

3. Remove and save the plunger from a 10 ml syringe and connect to the short end of the C18 column.

4. Pre-wet the C18 reversed-phase column with 5 ml of 100% acetonitrile

5. Equilibrate the column sequentially with three washes of 0.1% TFA using 1, 5, and 6 ml.

6. Load the acidified peptide sample onto the column; the peptides will bind to the C18 material.

7. Wash the column sequentially with three washes of 0.1% TFA using 1, 5, and 6 ml.

8. Wash the column with 2 ml of 5% acetonitrile with 0.1% TFA.

9. Place each column above new, clean 50 mL collection tube.

10. Elute the peptides 3 times with 3 ml of 40% acetonitrile with 0.1% TFA.

11. Eluted peptides should then be frozen completely to proceed to the lyophilization step. Freeze for 4-16 hours in -80°C freezer or on a dry ice and ethanol bath.

See specific PTMScan protocol for further details.

Description

C18 Reversed-phase solid phase extraction columns are useful tools for purifying and desalting peptides in preparation for PTMScan protocols and other mass spectrometry applications.

Background

Reversed-phase chromatography is an important technique for analytical and preparative biochemical separation and purification. Hydrophobic proteins, peptides, and nucleic acids can be separated by reversed-phase chromatography with excellent resolution and recovery. Furthermore, the use of ion pairing modifiers in the mobile phase allows reversed-phase chromatography of charged solutes such as hydrophilic peptides and fully deprotected oligonucleotides. Preparative reversed-phase chromatography has been used in applications ranging from micro-purification of protein fragments for sequencing (1) to process scale purification of recombinant protein products (2) and mass spectrometry proteomic analysis (3).

  1. Renlund, S. et al. Peptides 14, 1125-32.
  2. Olson, C.V. et al. (1994) J Chromatogr A 675, 101-12.
  3. Rush, J. et al. (2005) Nat Biotechnol 23, 94-101.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!


 

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