Affinity purification of C-terminus Avitag proteins using AbCA agarose

货号：

AbCA

Affinity purification of C-terminus Avitag proteins using AbCA agarose

A monoclonal antibody was developed that reacts only to the AviTag when
fused to the C-terminus of desired protein. It will identify C-terminus fusions
of AviTag on western blots and has been immobilized on agarose beads for
purifications purposes (catalog #AbC agarose). The AviTag containing
protein can be eluted from the monoclonal antibody affinity column using
documented mild elution conditions which retaining protein activity Elution
buffer I(Thompson NE, Hager, DA and Burgess, RR (1992) Biochemistry 31,
7003-7008; Thompson NE and Burgess, RR (1994) Protein Expression and
Purification 5,468-475). If protein can withstand elution at pH 3.0, Elution
buffer II can be used.
Reagents provided by Avidity, LLC
Affinity purified IgG against AviTag fused to C-terminus of disired protein
crosslinked to agarose 10 mg IgG/ml resin in PBS containing 0.05%
sodium azide
20 ml column for your use. Comes with upper bed support that prevents
column from drying out, column plug and cap.
Solutions: to be provided by user
PBS: 138 mM NaCl, 2.6 mM KCl in 10 mM potassium phosphate (pH 7.4)
PBST: PBS plus 0.05% Tween 20
Elution buffer I: 40% 1,2, propanediol, 0.75M Sodium pyruvate, 50 mM
Tris-HCl, 1 mM EDTA pH 7.5
Elution buffer II: 0.1 M glycine pH 3.0
Procedure
1. Affinity column material is poured into column and equilibrated with 20-40
ml PBS or acceptable buffer containing 100mM NaCl. Less ionic strength
buffers can be used but non-specific binding of protein will occur. Upper
bed support can be placed on top of resin to prevent accidental drying out
of column. Once bed support is in place, it is difficult to remove resin but
can be done. Column can also be ran without bed support with little
damage to the resin.
2. Prepare solution or extract containing protein of interest that is fused to
AviTag (C-terminal presentation). (Note 1)
AVIDITY LLC: 877.333.6063
3. The amount of extract to be circulated through the column depends on
the amount of protein being purified within the extract. Use at least 5 ml
of extract through the column. Collect flowthrough solution and reapply
to column 3-5 times to maximize binding sites of the column. This can be
done at RT or at 4oC. (Note 2)
Collect final Flow through of extract for evaluation by SDS-gel
4. electrophoresis or activity assay if possible.
5. Wash column with PBS until protein levels are not detected.
6. Apply 20 ml of selected elution buffer to column. Collect 0.5 ml fractions.
Evaluate fractions for protein concentration. If Elution buffer I is used,
spectrophotometric protein determination (A280) is not possible. We
suggest using Coomassie Plus Protein Assay Reagent (Pierce, #23236,
www.piercenet.com)
7. Pool fractions containing protein. Evaluate purity by SDS-gel
electrophoresis or activity assay if possible. (Note 3)
8. Dialysis of eluted protein is usually necessary for further use of purified
protein. Dialyze purified protein into appropriate buffer. (Note 4)
9. Typically, recovery of 1 mg of C-terminal AviTag fusion will be attained
when 2 mls of AbC agarose is totally saturated with desired protein. (Note
5)
Notes
1. Column will work when extracts are prepared using BPER following the
procedures outlined by Pierce. The detergents do not seem to hinder the
affinity of the MAB to C-terminal AviTag fusions.
2. Two ml of AbC agarose will typically purify 1 mg of C-terminal AviTag
fusion. Determine amount of extract based on this recovery. If extracts
contain more than 1 mg of desired protein, retain flow through solutions for
further purifications.