The Thermo Scientific Melon Gel Monoclonal IgG Purification Kit is a high-yield, mild purification system for monoclonal antibodies from ascites or hybridoma cell culture supernatant, or for large scale antibody purification from serum.
Unlike typical bind-and-elute affinity methods, Melon Gel Purification is based on the binding of non-IgG proteins from a prepared supernatant or ascites sample, thereby allowing the pure IgG to be recovered in the flow-through (non-bound) fraction. Antibodies are recovered ready for use in assays or subsequent conjugation and other modification steps. There is no need to neutralize or desalt to mitigate the effects of harsh elution buffers. Melon Gel Purification saves time compared to classical Protein A or Protein G methods without compromising the quality of the antibody.
- Fast – purify antibodies in half the time of required for Protein A or G
- Efficient – recovery and purity is often better than Protein A or G purification
- Robust – effective for subclasses of IgG that bind poorly to Protein A or G
- Gentle – antibodies retain activity because they are not exposed to denaturing conditions
- Regenerable – support can be regenerated and used for up to 6 purifications
- Compatible – amine-free buffer does not require removal or neutralization
- Flexible – accommodates a broad range of sample volumes and processing vessels
- Cell culture: spin columns can be used for purifications < 30mL of supernatant;
filter flasks can be used for bulk processing of samples up to 1L
- Ascites fluid: spin columns can be used for purifications up to 6mL
Simpler and faster than Protein A and Protein G purification. Side-by-side comparison of protocols for antibody purification of cell culture supernatant with the Thermo Scientific Melon Gel Monoclonal IgG Purification Kit and Protein A (or Protein G) agarose beads.
|Thermo Scientific Melon Gel
|Typical Protein A Column
- Concentrate supernatant to < 100mL, then exchange sample into Purification Buffer.
- Add sample solution to Melon Gel Resin.
- Incubate batch-wise for 5 minutes.
- Vacuum filter to recover purified IgG.
- Concentrate supernatant to < 100mL, then exchange sample into Binding Buffer.
- Add sample to packed and equilibrated column.
- Incubate sample with resin for 30 to 60 minutes.
- Wash column with 1 to 20 bed-volumes of buffer.
- Elute three times (minimum) with Elution Buffer.
- Neutralize IgG-containing sample.
|Total time: 2.1 hours
(no final dialysis needed)
|Total time: > 4 hours
(without subsequent dialysis)
The Thermo Scientific Melon Gel Kit provides better antibody recovery and purity from cell culture supernatant than Protein G. IgG was purified from cell culture supernatant containing 10% fetal bovine serum (FBS), resolved by SDS-PAGE and detected with Thermo Scientific Imperial Protein Stain (Part No. 24615).
Lane 1. Original cell culture supernatant sample.
Lane 2. Reconstituted pellet from precipitation with the Saturated Ammonium Sulfate Solution (Part No. 45216).
Lane 3. Melon Gel-purified sample.
Lanes 4-6. Protein G-purified samples.
The Thermo Scientific Melon Gel Kit provides better recovery and purity from ascites fluid than Protein G. Mouse IgG1 was purified from ascites fluid, resolved by SDS-PAGE and detected with Thermo Scientific GelCode Blue Stain Reagent (Part No. 24590). The Protein G-processed sample (Lanes 6-8) is contaminated with transferrin; the Melon Gel-purified sample pre-treated with Ascites Conditioning Reagent is not.
Lane 1. Original ascites fluid sample.
Lane 2. Sample treated with Ascites Conditioning Reagent (Part No. 45219) and purified over Melon Gel. Lane 3. Flow-through from Protein G column.
Lanes 4-5. Washes from Protein G column.
Lanes 6-8. Samples eluted from Protein G column.
|Thermo Scientific Melon Gel Support provides higher yields than Protein G purification. Anti-KLH IgG3 antibody was purified from cell culture media containing 10% FBS. Recovered antibodies were applied to a KLH-coated microplate. HRP-labeled goat anti-mouse antibody (Part No. 31430) and TMB substrate (Part No. 34028) were used to detect the bound antibodies. Absorbances at 450nm of the purified samples were compared. Recoveries of antibodies produced in serum-free media and ascites fluid were also measured similarly and found to be greater than or equal to recoveries obtained with Protein G Agarose.
Specialized protocols for culture supernatant and ascites fluid:
The instructions for the Melon Gel Monoclonal IgG Purification include separate protocols for cell culture supernatant samples and ascites fluid samples. For the latter protocol, Ascites Conditioning Reagent (Part No. 45219) is required.
Accessory products for the Melon Gel Monoclonal IgG Purification Kit.
|Cell Culture Supernatant
||Part No. 45216
Saturated Ammonium Sulfate Solution (1L)
|Optional: Provides for highest possible purity, application of more antibody per milliliter of Melon Gel Resin and a greater number of regeneration and reuse cycles
||Part No. 45219
Ascites Conditioning Reagent , 5mL
|Required: Precipitates transferrin and other ascites fluid components that interfere with Melon Gel Purification
Other IgG purification kits and package sizes of Melon Gel Resin are available. All sizes use the same formulation of Melon Gel Resin, Purification Buffer and Regenerant Solution but have slightly different protocols based on the most common application for each package size. Components of any package size can be used at an appropriate scale with any one of the procedures.
Melon Gel Monoclonal IgG Purification Kit FAQ
Review of antibody purification methods
Melon Gel IgG Purification Kits (for serum samples)
Protein A, Protein G and other IgG purification resins
Purification products for IgM, IgA, IgY