Product Information
Product Usage Information
Notes:
• Approximately 20 mg of protease digested peptides can be purified on one C18 reversed-phase column.
• The steps described in the Directions for 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.
Storage
Product Description
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).
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Cross-Reactivity Key
H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected
Trademarks and Patents
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