| Starting Material: |
Crude extract from 100 ml culture. |
| Step 1: |
Pre-clear crude extract
Pass material through 1mL Sepharose column to remove any proteins
that non-specifically bind to Sepharose.
Affinity Matrix Preparation
The supplied affinity matrix was prepared by cross-linking 2mg
of purified monoclonal anti-epitope tag antibody to 1mL of NHS-activated
Sepharose. |
| Step 2: |
Equilibrate Affinity Column
Transfer the affinity matrix to a column and equilibrate with
Column Buffer* containing 0.05% Tween 20 or equivalent and 0.5M
NaCl at 4C. |
| Step 3: |
Load pre-cleared material and wash
Load the sample onto the affinity column and re-apply the flo-thru
in order to achieve quantitative binding. After loading the sample,
wash with 100mL of Column Buffer* containing 0.5M NaCl and 0.05%
Tween 20 or equivalent. |
| Step 4: |
Elution
Prepare an elution buffer by dissolving 1mg of epitope tag peptide
in 2.5mL of Column Buffer. Apply the elution buffer to the column,
incubate for 5-15 minutes, then collect fractions. Pool peak
fractions. Wash column with 3mL Column Buffer*.
Other elution buffers such as 0.1M glycine, pH 2.8, or 40mM
diethyl-amine, pH 11.0 may be used. Elution volumes may be optimized
depending on amount of bound material, column volume, etc. |
| Step 5: |
Wash & Regenerate Column
Wash column with 100mM glycine buffer, pH 2.9 followed by Column
Buffer* containing 0.5M NaCl and 0.05% Tween 20 or equivalent. |
| Step 6: |
Concentrate Pooled Fractions (if desired) |
| Step 7: |
Storage of Column
The column should be stored at 4C in PBS containing 0.03%
Thimerosal. |
|
*Column Buffer
200mM MES 2-(N-morpholino)ethanesulfonic acid, pH 6.2
(or equivalent such as Tris, phosphate, or other common buffers)
0.1mM MgCl2 0.1mM EDTA 1mM beta-mercaptoethanol (optional, >2mM
risks reducing antibodies on the matrix) 1mM phenylmethylsulfonyl
fluoride (PMSF) or other protease inhibitors of choice |
