How does Melon Gel Purification work?
At physiological pH Melon Gel Support binds non-antibody proteins in serum, such as albumin and transferrin, allowing the antibody to flow through in a mild buffer suitable for storage and downstream applications.
What formats are available for the Melon Gel Support?
There are four different formats. The Melon Gel IgG Purification Kit (#45212) and Melon Gel IgG Spin Purification Kit (#45206) are for purifying antibodies from serum. The Melon Gel Monoclonal IgG Purification Kit (#45214) is for purifying monoclonal antibodies from cell-culture supernatant and ascites fluid. Ready-to-use prepacked chromatography cartridges are available as two 1 ml columns (#89932) or one 5 ml column (#89933).
Does ascites fluid require any special treatment before processing?
Yes, ascites samples must be treated with the Ascites Conditioning Reagent (#45219) before antibody purification. See the instructions and other product information for the Monoclonal IgG Purification Kit (#45214).
Are columns provided in the kits?
Microcentrifuge spin cups are provided in the Melon Gel IgG Spin Purification Kit (#45206). No columns are included in the other Melon Gel Kits (#45212 and #45214) so that researchers can adjust the scale of their purification. A wide variety of disposable columns are available for use with these kits.
Can the Melon Gel Method remove BSA or gelatin from commercially available antibody?
Yes. Dialyze or desalt the antibody solution against the Purification Buffer provided in the kit. The undiluted antibody solution can then be added to the Melon Gel Support using the volumes indicated in the instructions. Tech Tip #55 provides a protocol for this method.
After purification, must I desalt or dialyze the purified antibody before using the Pierce Direct IP Kit (#26148)?
No. However, researchers may want to add an equal volume of binding buffer to elevate the pH towards neutral.
How many times can I reuse the Melon Gel Support?
The Melon Gel Support can be used up to three times. Performing an ammonium sulfate precipitation on serum samples containing large amounts of non-antibody protein before purification improves purification efficiency and increases reusability.
Is Melon Gel Purification a viable alternative to Protein A and Protein G?
Yes. 90% recovery of IgG and greater than 80% purity are typically observed for a single purification. Although IgA and IgM may be present with the purified IgG, a high percentage (> 80%) of IgA and IgM binds to the support. This high binding combined with the fact that IgA and IgM are present in low abundance in serum results in low levels of these components in the purified IgG.
Can Melon Gel purify chicken IgY?
No. Pierce Thiophilic Adsorbent (#20500 and #44916) can be used to purify IgY from serum. The Chicken IgY Purification Kit (#44918) can be used to purify IgY from egg yolks.
How can I determine when the Melon Gel Support is saturated?
For serum, a volume double the Melon Gel Support volume is the maximum amount that can be processed at one time. To determine if too much sample was processed, examine the purified IgG by SDS-PAGE for additional protein bands.