Thermo Scientific Pierce NHS-Activated Agarose is a high-quality, amine-reactive, beaded-agarose resin for rapid and stable immobilization of proteins, peptides and other ligands via primary amines. NHS-activated Sepharose 4 Fast Flow is described separately (refer to Data file 18-1113-53). Sepharose 4 Fast Flow matrix. Sepharose 4 Fast Flow is a highly cross-linked agarose matrix. In its preactivated CNBr form, it offers much improved performance when compared with the well-established CNBr-activated Sepharose 4B. Mechanism of activation of Sepharose and Sephadex by cyanogen bromide Joachim Kohn and Meir Wilchek* Department of Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel (Received 29 September 1981; revised 4 November 1981) The mechanism of CNBr activation of polysaccharide resins like Sepharose and Sephadex has been elucidated using recently published analytical procedures for. NHS-activated Sepharose 4 Fast Flow Ligand density 16–23 µmol NHS/ml drained gel pH stability** long term 2–13 short term (CIP) 2–13 Table 1. Characteristics of NHS-activated Sepharose 4 Fast Flow. * At 25 °C in water in an XK 50/60 column, 25 cm bed height. The flow rate after coupling may differ depending on the ligand. ** Depends on the ligand. CNBr-activated Sepharose will react with Tris buffer. It is important to remove any traces of Tris from the antibody solution.
Methods Enzymol. Author manuscript; available in PMC 2015 Feb 23.
Published in final edited form as:
doi: 10.1016/B978-0-12-420119-4.00003-3
NIHMSID: NIHMS660670
The publisher's final edited version of this article is available at Methods Enzymol
See other articles in PMC that cite the published article.
Associated Data
NIHMS660670-supplement-Supplementary_Data.pdf (199K)
Abstract
Antibodies will be immobilized on a cyanogen bromide-activated Sepharose for subsequent use in pull-down assays or immunoaffinity purification (see Immunoaffinity purification of proteins).
1. THEORY
Cyanogen bromide (CNBr)-activated Sepharose is a readily available commercial product. Proteins are coupled to the resin through primary amines. While other coupling chemistries are available, the CNBr-based resin is a good choice because of the mild reaction conditions and broad applicability to different types of proteins.
2. EQUIPMENT
- Centrifuge
- Nutator mixer or rocking platform mixer
- UV/vis spectrophotometer
- Magnetic stir plate
- Beaker, 1 l
- Magnetic stir bars
- Dialysis tubing or Slide-A-Lyzer dialysis units
- Amicon protein concentrators (optional)
3. MATERIALS
- Purified monoclonal antibody
- CNBr-Activated Sepharose 4 Fast Flow (GE Healthcare)
- Hydrochloric acid (HC1)
- Sodium bicarbonate (NaHCO3)
- Sodium chloride (NaCl)
- Tris base
- Sodium acetate (NaOAc)
- Potassium chloride (KCl)
- Sodium phosphate monobasic (NaH2PO4)
- Potassium phosphate, dibasic (K2HPO4)
- Sodium carbonate (Na2CO3)
3.1. Solutions & buffers
Step 1 Activation buffer.
Dilute 4 μl HCl in 50 ml water for a final concentration of 1 mM |
Coupling buffer.
Component | Final concentration | Stock | Amount |
NaHCO3, pH 8.3 | 100 mM | 1 M | 100 ml |
NaCl | 500 mM | 5 M | 100 ml |
Add water to 1 l |
Step 3 Quenching buffer.
Dilute 5 ml of 1 M Tris–HCl, pH 8.0 in 45 ml water for a final concentration of 100 Mm |
Step 4 High pH wash buffer.
Component | Final concentration | Stock | Amount |
Tris–HCl, pH 8.0 | 100 mM | 1 M | 25 ml |
NaCl | 500 mM | 5 M | 25 ml |
Add water to 250 ml |
Nhs Activation Of Sepharose 6b 5
Low pH wash buffer.
Component | Final concentration | Stock | Amount |
NaOAc, pH 4.0 | 100 mM | 1 M | 25 ml |
NaCl | 500 mM | 5 M | 25 ml |
Add water to 250 ml |
Storage buffer (PBS, pH 7.4)
Component | Final concentration | Stock | Amount |
NaCl | 137 mM | 5 M | 1.37 ml |
KCl | 2.7 mM | 4 M | 33.8 μl |
NaH2PO4 | 10 mM | 0.5 M | 1 ml |
K2HPO4 | 2 mM | 0.5 M | 0.2 ml |
Add water to 50 ml |
4. PROTOCOL
4.2. Preparation
Obtain purified monoclonal antibody. The antibody can be purchased commercially or purified from either ascites fluid or media from hybridoma cell lines.
See Fig. 3.1 for the flowchart of the complete protocol.
Flowchart of the complete protocol, including preparation.
5. STEP 1 PREPARATION OF ANTIBODY AND RESIN
5.1. Overview
Dry resin is swelled and activated. The antibody is dialyzed into a buffer compatible for coupling.
5.2. Duration
4–5 h
- 1.1 Dialyze the antibody into cold Coupling Buffer at 4 °C. Change to fresh buffer after 2 h and continue dialyzing for another 2 h.
- 1.2 Measure the absorbance at 280 nm of the final antibody solution and calculate its concentration.
- 1.3 Concentrate the antibody to 1–2 mg ml−1 if it is too dilute.
- 1.4 Determine the amount of resin needed. Approximately 2 mg of antibody can be coupled to 1 ml of swollen resin.
- 1.5 Weigh out 0.25 g of dry resin for every 1 ml of hydrated resin needed.
- 1.6 Add 5-column volumes of cold Activation Buffer to resin.
- 1.7 Incubate on a nutator or platform rocker for 2 h at 4°C.
5.3. Tip
CNBr-activated Sepharose will react with Tris buffer. Download and install mysql in windows. It is important to remove any traces of Tris from the antibody solution.
5.4. Tip
Performing the reaction with antibodies in a buffer other than Coupling Buffer will reduce coupling efficiency.
5.5. Tip
Coupling efficiency is maximized when the antibody is at a final concentration of 1–2 mg ml−1. A 1 mg ml−1 solution of antibody usually will have an OD280 = 1.4.
5.6. Tip
The ratio of antibody to resin can be varied as needed for downstream applications.
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See Fig. 3.2 for the flowchart of Step 1.
Flowchart of Step 1.
6. STEP 2 COUPLING THE ANTIBODY TO THE RESIN
6.1. Overview
The antibody is chemically coupled to the resin.
6.2. Duration
Overnight
- 2.1 After swelling the resin, centrifuge it at 1000 × g for 5 min. Decant the supernatant.
- 2.2 Add dialyzed antibody to the resin and incubate overnight on a nutator at 4 °C.
6.3. Tip
Extra CNBr resin can be prepared and incubated with coupling buffer lacking antibody to generate a negative control in later applications to test nonspecific binding to the CNBr support.
See Fig. 3.3 for the flowchart of Step 2.
Flowchart of Step 2.
7. STEP 3 QUENCH THE REACTION
7.1. Overview
Wash any unreacted antibody from the resin and then ensure that there are no unreacted CNBr sites remaining.
7.2. Duration
4 h
- 3.1 Centrifuge the resin at 1000 × g for 5 min.
- 3.2 Remove the supernatant and save.
- 3.3 Measure the OD280 of the supernatant.
- 3.4 Add 5 column volumes of Coupling Buffer to the resin.
- 3.5 Incubate on nutator mixer for 30 min at room temperature.
- 3.6 Spin down resin at 1000 × g for 5 min and decant supernatant.
- 3.7 Add 5–10 column volumes of Quenching Buffer.
- 3.8 Incubate on nutator for 2–3 h at room temperature.
- 3.9 Spin down the resin at 1000 × g for 5 min and decant supernatant.
7.3. Tip
The coupling efficiency can be calculated by dividing the total amount of antibody in the supernatant in Step 3 by the total amount of antibody loaded on the column in Step 2. Typical coupling efficiencies are in the range of 70%.
See Fig. 3.4 for the flowchart of Step 3.
Flowchart of Step 3.
8. STEP 4 WASH THE RESIN
8.1. Overview
Remove uncoupled antibody from the resin and prepare resin for long-term storage.
8.2. Duration
4 h
- 4.1 Resuspend the resin in 10 column volumes of High pH Wash Buffer.
- 4.2 Spin down the resin at 1000 × g for 5 min and decant supernatant.
- 4.3 Resuspend the resin in 10 column volumes of Low pH Wash Buffer.
- 4.4 Spin down the resin at 1000 × g for 5 min and decant supernatant.
- 4.5 Repeat Steps 4.1–4.4 two more times.
- 4.6 Resuspend the resin in 5 column volumes of Storage Buffer.
- 4.7 Spin down the resin at 1000 × g for 5 min and decant supernatant.
- 4.8 Add 1 column volume of Storage Buffer and store resin at 4 °C.
See Fig. 3.5 for the flowchart of Step 4.
Flowchart of Step 4.
Supplementary Material
Supplementary Data
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