| 1IgG |
|---|
Analyte not found
Applications
IgG Dog Reduced Jupiter C4 300
14909
Separation Mode: Reversed Phase
Reversed Phase
C4
HPLC
Pharmaceutical/Biopharmaceutical
Life Science
Protein
Biochemical Compound / Nutrient
Analytes
Details
LC Conditions (App ID: 14909)
Column: | ||||||||||||
Brand Name: | ||||||||||||
Part No: | ||||||||||||
Phase Name: C4 | ||||||||||||
Elution Type: Gradient | ||||||||||||
Mobile Phase: A: 0.1% TFA in Water/Acetonitrile (95:5) B: 0.085%TFA in Acetonitrile/IPA/Water (75:20:5) | ||||||||||||
Gradient Profile:
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Flow Rate: 0.25 mL/min | ||||||||||||
Temperature: 22°C | ||||||||||||
Detection: UV-Vis Abs.-Diode Array (PDA) @ 220 nm (22°C) | ||||||||||||
Sample Note: Application Focus: Reducing antibodies to separate heavy and light chains by Jupiter C4
While using IPA in the organic mobile phase can help improve the chromatography of Ig-G, often the best way to observed changes in immunoglobins is by reducing the proteins and separating the heavy and light chain of the protein and analyzing them by HPLC on a Jupiter C4 column. Key to get complete reduction of a protein is to perform the reduction in a chaotrope (either urea or guanidine) to unfold the protein thus breaking up any non-covalent interactions. Upon incubation with DTT, BME or some other reducing reagent, Ig-G heavy and light chains are released and can by separated by HPLC. In this example, dog Ig-G was reduced with 2 mM dithiothreitol in 8 M Urea at 45º C for 30 minutes.
In this example dog Ig-G was reduced with dithiothreitol and injected on a Jupiter 300 C4 column. As shown in App ID# 14909, one can see the heavy and light chains are baseline resolved using the conditions discussed in length in App ID#14908 (20% IPA in the organic mobile phase and elevated temperature). These results demonstrate the ability of the Jupiter 300 C4 to resolve differences between large hydrophobic proteins for demanding applications such as the separation of immunoglobulin proteins. |
Order Items Used in This Application
00F-4167-B0
Jupiter 5 µm C4 300 Å, LC Column 150 x 2 mm, Ea
Qty:
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