Melon Gel Filer

Melon Gel Monoclonal IgG Purification Kit ™

It’s almost as easy as having someone else do the work for you!

The new Melon™ Gel Monoclonal IgG Purification Kit from Pierce offers a fast and simple protocol for purifying IgG from cell culture supernatants and ascites that renders Protein A and Protein G methods obsolete How does Melon™ Gel work?

Highlights:

Melon™ Gel is composed of a proprietary combination of ligands that retains the component proteins typically found in serum and allows IgG to pass through.

• Fast – purify antibodies in half the time of Protein A or Protein G

The Melon™ Gel Monoclonal IgG Purification Kit:

• Efficient – recovery and purity is often better than Protein A or Protein G purification • Robust – effective for antibodies known not to bind to Protein A or Protein G

• Replaces tedious bind-and-release antibody-isolation methods, resulting in a fast, easy and gentle approach to antibody recovery and purification

• Gentle – antibody retains activity because it is not exposed to denaturing conditions

• Delivers monoclonal antibodies (MAbs) with quality and purity equal to – or better than – that obtained using classical Protein A or Protein G purification methods

• Compatible – buffer is amine-free and does not require removal or neutralization

• Purifies monoclonal IgG from up to 1 L of cell culture supernatant (serum-containing or serum-free) and up to 200 ml of ascites fluid

• Regenerable – support can be used for up to six purifications

• Flexible – accommodates a broad range of sample volumes Cell culture: Spin columns can process supernatant samples that are < 30 ml. Filter flasks allow processing of samples up to 1 L. Ascites: Spin columns process samples up to 6 ml.

Figure 1. Melon™ Gel offers better 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 Imperial™ Protein Stain† (Product # 24615). Lane 1: Original cell culture supernatant, Lane 2: IgG after precipitation with Saturated Ammonium Sulfate (Product # 45216), Lane 3: Melon™ Gel-purified IgG and Lanes 4-6: Protein G-purified IgG.

www.piercenet.com/melg92h

Figure 2. Melon™ Gel offers better recovery and purity from ascites fluid than Protein G. IgG1 was purified from ascites fluid, resolved by SDS-PAGE and detected with GelCode™ Blue Stain Reagent (Product # 24590). Lane 1: Original ascites fluid, Lane 2: treated with Ascites Conditioning Reagent (Product # 45219) and purified with Melon™ Gel, Lane 3: Protein G column flow-through, Lanes 4-5: Protein G column washes and Lanes 6-8: Protein G column elutions. NOTE: The Protein G-purified sample is contaminated with transferrin (Lanes 6-8). The Melon™ Gel-purified sample is not (Lane 2).

Cell Culture Supernatant Purification

3

1. Buffer exchange 2 x 1 hour. U.S. 5,503,741 # 5,503,741 Pat. # U.S. Pat.

4

Slide-A-Lyzer®

1

2. In a funnel, drain storage buffer from resin and then transfer funnel to a new filter flask. Add buffer-exchanged sample and stir for 5 minutes.

Cassette

4

0.40 Absorbance at 450 nm

U.S. 5,503,741 # 5,503,741 Pat. # U.S. Pat.

5. Centrifuge to collect purified sample.

Ordering Information

0.45 0.35

DESCRIPTION

45214

Melon™ Gel Monoclonal IgG Purification Kit† Kit Sufficient reagents to purify IgG from up to 1 L of cell culture supernatant or up to 200 ml of ascites fluid. Includes: Melon™ Gel IgG Purification Support, 200 ml settled gel Melon™ Gel Purification Buffer, 100X, dry mix Melon™ Gel Regenerant, 5X, dry mix

45216

Saturated Ammonium Sulfate Solution For use with the Melon™ Gel Monoclonal IgG Purification Kit and Melon™ Gel IgG Purification Kits for Sera or as a general-purpose salting-out agent for protein-preparation applications.

45219

Ascites Conditioning Reagent 5 ml For use with the Melon™ Gel Monoclonal IgG Purification Kit. Instructions not included. Use of this reagent is described in the instructions that are provided with Product # 45214.

0.25 0.20 0.15 0.10

Melon™ Gel Protein G

0.05 0.00 1.00

2.00

3.00

4.00

Amount of KLH (µg/well)

Figure 3. Melon™ Gel provides greater IgG recovery than Protein G. AntiKLH IgG3 produced in cell culture was purified using Melon™ Gel and Protein G agarose. Recoveries were determined by applying the Melon™ Gel- and Protein G-purified antibodies to KLH-coated plates and detecting with HRP-labeled goat anti-mouse antibody (Product # 31430) and 1-Step™ Ultra TMB-ELISA (Product # 34028). The absorbances of the purified samples were compared. Recovery of antibodies produced in serum-free media and ascites fluid were also greater than or equal to recoveries obtained with Protein G.

PKG. SIZE

PRODUCT #

0.30

0.00

3. Remove Ascites Conditioning Reagent via dialysis or desalting.

4. In a spin column, combine buffer-exchanged sample with gel and incubate end-over-end for 5 minutes.

3. Apply vacuum to collect the purified sample in the new flask.

2. Centrifuge sample at 5,000 x g for 10 minutes.

3

1

1. Combine the Ascites Conditioning Reagent with purification buffer and then add to the sample. Mix end-over-end for 10 minutes.

2

Cassette

2

Slide-A-Lyzer®

Ascites Purification

†

1L

U.S. patent pending on Melon™ Gel Monoclonal IgG Purification Technology and Imperial™ Protein Stain Technology.

Tel: 815-968-0747 or 800-874-3723 • Fax: 815-968-7316 • Customer Assistance E-mail: [email protected] • www.piercenet.com © Pierce Biotechnology, Inc., 2005. Pierce products are supplied for research or manufacturing applications only.

# 1601296 09/05