immunoaffinity chromatography
immunoaffinity chromatography
This experiment introduces the method of immunoaffinity chromatography. This experiment is from the Laboratory Guide for Protein Purification and Identification, by Zhu Houzhu.
Operation method
immunoaffinity chromatography
Materials and Instruments
Immunoaffinity media Cyanogen bromide (CNBr) Activated Sepharose 4B HCl 55% saturated AMS precipitates Coupling buffer Ethanolamine buffer B LAC Elution buffer Dithiothreitol Potassium thiocyanate Sodium azide Move Materials and equipment For more product details, please visit Aladdin Scientific website.
Econo-column SDS-PAGE Electrophoresis unit
Immunoaffinity media (with immobilized MAb NT73; see below)
Cyanogen bromide (CNBr) activated Sepharose 4B (Sigma Chemical Co.C 9142)
HCl (1 mmol/L)
55% saturated AMS precipitate (see p. 154)
Econo-column (Bio-Rad Laboratories)
SDS-PAGE Electrophoresis Unit
Reagents
Coupling buffer
Ethanolamine (1 mol/L)
Buffer B
LAC elution buffer
Dithiothreitol (DTT) (0.1 mol/L)
Potassium thiocyanate (KSCN)(2mol/L)
Sodium azide (2%)
(For formulas, see "Preparation of Reagents pp.l84~189)
Operating Procedures
Preparation and storage of immunoaffinity media
The MAb-producing mouse hybridomas used in this procedure were prepared according to standard methods (see Harlow and Lane, 1988) MAb's were precipitated with 45% saturated AMS and purified from mouse ascites by taking advantage of their inability to bind to DEAE-fibre columns at pH 7.0, 25 mmol/L NaCl. Under these conditions, many mouse IgG MAb can be effluxed through DEAE columns, and the resulting MAb preparations are >80% pure and are suitable for immunoaffinity chromatography (see Harlow and Lane, 1988, for many of the methods used to purify MAb. MAb can be covalently coupled to Sepharwse activated with cyanogen bromide (CNBr) by the methods outlined below. See also Pharmacia publication Affinity Chromatography: Principles and Methods (Pharmacia LKB Biotechnology, 1993).
1) Take 1 g of CNBr-activated Sepharose4B gel (volume about 3.5 ml), dissolve it in 1 mmol/LHCl and wash it.
2) Mix about 10 mg of MAb dissolved in coupling buffer with the washed Sepharose 4B gel for 2 h at room temperature.
3) Place the Sepharose 4B gel in 1 md/L ethanolamine for 2 h at room temperature to seal the remaining reactive groups on the gel.
4) Wash off excess protein and sealer. Under normal conditions, each ml of immunoaffinity medium contains 2-3 mg of covalently coupled MAb. The immunoaffinity medium is stored at 4°C in buffer containing 0.02% sodium azide to prevent bacterial growth.
Immunoaffinity Chromatography
In this experiment, an immunoaffinity column was used to bind core RNA polymerase and various bound σ factors. Since σ 32 is overproduced in the departing cells, most core RNA polymerases contain bound σ 32 (under normal conditions, σ 32 may be only a minor component of the RNA polymerase). The core RNA polymerase-σ 32 complex is acidic and can be used for
The core RNA polymerase-σ32 complex is acidic and can be precipitated with PEI.
1) Resuspend 2 ml of Immunoaffinity Media Slurry in 10 ml of Buffer B before use. Centrifuge at 1000r/min for 1-2 min at room temperature, gently, as the force of centrifugation at high speeds can crush the gel. Repeat this step once. Repeating this step once will remove sodium azide and MAb leached from the gel during storage.
2) Dissolve 55% of the saturated AMS precipitate in 20 ml of Buffer B. Centrifuge at 13,000 r/min for 10 min at room temperature to remove all insoluble material (and the amount of precipitate, if any, should be minimal). The supernatant is retained and sampled (Sample I).
3) Add the dissolved, clarified AMS precipitate to the precipitated Sepharose 4B gel and mix for 30 min at room temperature with gentle shaking.
Additional protocol experiments: 100 ul of each sample is taken at 0, 5, 15, and 30 min. Each sample is immediately centrifuged and the supernatant analyzed by SDS-PAGE to determine how long it takes for proteins to bind to the immunoaffinity medium (see p. 178).
Note: The contact with the immunoaffinity medium must be with a buffer containing only a low concentration of reducing agent (DTT), otherwise the disulfide bonds of immunoglobulin G (IgG) will be reduced, resulting in the detachment of the IgG heavy and light chains from the medium. For this reason, we use Buffer B (without DTT). Although RNA polymerase survives briefly in buffer without DTT, DTT should be added back to the column chromatography fractions as soon as possible. This is easily accomplished by preloading lul of 0.1mol/L DTT into each empty component tube. Remove 5% of the glycerol from Buffer B; if glycerol is present, some of the enzyme will elute from the column during the wash due to the polyol-responsive nature of the MAb used in this step.
4) Slowly centrifuge the chromatography medium, discard the supernatant (IAC pass-through), and resuspend the medium in 10 ml of Buffer B before injecting into the Bio-Rad Econo column.
5) Collect 10 ml of pass-through solution (1st wash), add 5 ml of Buffer B above, and collect another 5 ml of pass-through solution (2nd wash).
Note: Washing of the media and elution of the σ-factor from the media can be performed in batch mode in a test tube by adding the appropriate buffer, mixing slowly for 15-30 min, and then centrifuging the media slowly. Although the batch approach is more effective in binding the a32 factor to the medium and removing unbound material, subsequent washing and elution are performed on a single column. When the medium is placed in elution buffer, σ32 dissociates from the antibody, but must diffuse out of the 100um Sepharose beads in a three-dimensinal random walk, which takes a long time. Once σ32 is "walked" off the beads, it elutes from the beads more efficiently if the buffer is flowing from the side in column mode chromatography.
6) Add lml of IAC elution buffer to the column 10 times at room temperature and collect each fraction in lul of 0.1mol/L DTT in Eppendorf tubes. Fractions (1~10 tubes) were placed on ice and analyzed by rapid protein spot blot (Westerndotblot) or SDS gel electrophoresis to determine which tubes could be combined.
NOTE: The density of the IAC Elution Buffer is greater than the immunoaffinity media in Buffer B. The density of the IAC Elution Buffer is greater than the immunoaffinity media in Buffer B. (It may damage the column media upwelling when added!) . Therefore, it is important to allow the top of the column media to press down slightly at the end of the wash, and then add the first 1-ml of Elution Buffer very slowly (preferably 200ul first, allow it to sink, and then add the rest of the elution buffer slowly). If the flow rate of the column slows down when viscous elution buffer is added, a 6-10 cm long plastic tube can be connected to the bottom of the column to increase the hydrostatic pressure and encourage the buffer to flow through the column.
7) After the column has been used, treat the column with 2 mol/L KSCN (a strong denaturing salt with a high leaving sequence that does not irreversibly inactivate antibodies) to remove residual non-covalently bound proteins. The column is then washed with Buffer B and stored at 4°C in Buffer B containing 0.02% sodium azide. If the columns are carefully cleaned and stored, they can be reused dozens of times. 
POROS 50 Q Anion Exchange Chromatography (optional)
To obtain higher purity core RNA polymerase-σ32 complexes, the peak fractions of the IAC column can be diluted 10-fold with 50 mmol/L Tris (pH 7.9), reducing the propylene glycol to 3% and the NaCl concentration to 0.075 mol/L. Diluted samples can also be subjected to ion-exchange chromatography as described in PP.149-151, except that the FOROS 50S column is replaced by the POROS 50Q column. The diluted sample can also be subjected to ion exchange chromatography as described in PP.149~151, except that a POROS 50Q column is used instead of a FOROS 50S column. The final combined fractions are dialyzed against storage buffer (Buffer A + 50% glycerol) as described in P.151. The final combined fractions are dialyzed against storage buffer (Buffer A + 50% glycerol) as described in P.151.