Immobilised Metal-ion Affinity Chromatography (IMAC)

Metal Chelate Agaroses

His tag, consisting of five to ten Histidine residues, has been used for several years for purification of recombinant proteins by immobilised metal-ion affinity chromatography (IMAC).

The use of the His tag has several advantages, there is minimum addition of extra amino acids to the recombinant protein and the small Histidine tail is weakly immunogenic and does not interfere with protein folding.

His tagged proteins have a high affinity for metal ions. Nickel, Copper, Cobalt all attach His tagged proteins with varying degrees of affinity.

Affinity based capture methods have become routine techniques for isolation and purification of recombinant proteins and the IMAC methods offer a quick one step purification method.

The use of Nickel and Metal Chelate Agarose beads is specific in its affinity for clustered Histidine tagged proteins. The purified protein is bound to the gel in a neutral loading buffer and eluted using 250mM imidazole elution buffer.

The gel purification protocol enables the binding and elution of His tagged proteins in time efficient method, to aid identification by western blotting, LC-MS / Maldi-Tof or PAGE.

The Ni, Cu and Co agarose beads can be designed to suit the application as the three different forms work similarly offering, a degree of selectivity in the choice of affinity material to be used.
 

Product

Nickel II- Chelate IDA Agarose Gel. (Ni-II Chelate)

AB310-02 20ml Nickel chelate agarose

Typical binding capacity up to 20mg/ml gel*
Cross linked 6% agarose
Virtually no metal leaching

2ml Gel example in the following
anti-microbial storage buffer solution:

20% Ethanol,
10mM HEPES
50mM NaCl
pH 7.5
 

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Catalogue Numbers of Thio-Link Gel™ Kit

[Thio-Link Gel™ is supplied as a slurry in storage buffer (10mM EDTA-Na, 0.05% sodium azide, 50% glycerol) and the volumes quoted are the gel bed-volumes when settled in a column and ready for use.]

Catalogue Numbers of Ancillary Reagents for producing affinity purified anti-peptide antibodies.

Procedure for the purification of Anti-peptide Antibodies.

Please read instructions thoroughly before use.
(See Schematic diagram)

Step 4)
Attach the end cap to the bottom of the column and then, add the 1ml solution of Blocking Reagent to the column and thoroughly mix as before. The solution should be left to react for 30-60minutes at room temp.
(The schematic represents blocked off sites with an –X.)

Wash the column with 12mls of Coupling buffer (AB102-02) to remove excess blocking reagent and then wash the column with 20mls Pre-conditioning Buffer (AB108-01) to condition the column.

At this stage the column can be stored for later use. In order to store the column it needs to be washed with 10ml of Storage Buffer (AB106-02) which contains the preservative Thimerosal. 
Finally push the second frit supplied into the column, positioning it to rest above the gel, being careful not to compress the gel bed. Replace the end caps and store at 4oC.

Step 5)
Affinity Purification
Immediately before commencing Affinity Purification, it is important to both pre-condition the column and to pre-cycle it with acidic Elution Buffer (AB103-02) that will be used to Elute the antibody from the column. 
Wash the column again with 12ml of Preparation and Wash Buffer (AB105-02).
Follow this with 12ml of Pre-conditioning Solution (AB108-01) then wash with 12ml of Elution Buffer (AB103-01).
Finally wash the column with 24ml Preparation and Wash Buffer (AB105-02).

The column is now ready, to affinity purify a small sample (2ml) of the serum.
It is usual to dilute the sera 1in 4 with Prep & Wash Buffer (AB105-02). Pass the serum through the column several times to ensure maximum binding of the antibody to the column.

A small peristaltic pump can be used to re-circulate the serum through the column. 

Step 6)
The affinity-bound antibody can then be eluted or purified from the column by adding 10 x 1ml aliquots of Elution Buffer (AB103-02). Collect the antibodies in 1ml fractions in tubes containing 100 micro-litres of Neutralising Buffer (AB 107-01) and mix each fraction as it is collected. This protects the eluted antibody.
Carry this step out quickly as possible so the antibody is not exposed to acidic conditions for longer than necessary.

The antibody will usually start to emerge in fraction 5 with a maximum quantity in fraction 7.

Quantification
The absorbance of the fractions measured at 280nm will give a quantitative measure of the amount of antibody in the fractions.
Use 1ml of Elution Buffer (AB103-02) with 100 micro-litres of Neutralising Buffer (AB107-01) as a Blank.

Standardisation can be carried out using either BSA or Ig.G

Storage
Store the purified antibody at -20oC or -70oC after adding 20 micro-litres per ml of Thimerosal stock solution ( 250milli-grams per 100ml Water ) to each fraction.


Do not attempt to concentrate the antibody or freeze dry it as both procedures are likely to greatly reduce the antibody activity.

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Hydrazide Gel

Severn-Link Hydrazide Agarose

The Severn-Link Hydrazide Gel is a high efficiency glycoprotein binding medium that binds only to oxidised sugar molecules in the glycosyl groups; it does not bind to amino acids. Some examples of glycoproteins successfully coupled to a hydrazine support are: Collagen type VI, Human Ig G, Avidin, Chorionic Gonadotropin, Fetuin, Ovalbumin, Α1-acid glycoprotein and Pepsin.

Perhaps the most valuable use for Severn-Link Hydrazide Gel is with antibodies.
Iimmunoglobulins are glycosylated away from the hyper-variable region, therefore linking them to a solid support through oxidised glycosyl groups,which preserves full antibody functionality and integrity, which no other method of linking can guarantee.
The method employs the oxidation of sugar molecules with sodium meta-periodate converts vicinal pairs of hydroxyl groups to aldehyde groups, (-CHO), and it is these groups that bind to the hydrazide agarose as shown in the diagram below.

As antibodies are irreversibly bound to the matrix the column can now be used for affinity purification of the antigen or secondary antibody.

Alternatively if the antigen is a glycoprotein the bound glycoprotein can be used for affinity purification of antibodies.

Contents of Severn-Link Hydrazide Gel Kit
Catalogue Numbers

AB98 Whole kit includes 10ml of gel

Individual constituents of kit

AB200 Severn-Link Hydrazide Gel. (10ml).
AB101-00 10ml plastic tubes for making Severn-Link Hydrazide Gel columns. (10 Pack)
AB202-01 Agent for oxidising glycoproteins for attachment to the gel. (5x5mg Sodium meta-Periodate).
AB204-01 Storage Buffer, (PBS pH 7.4 with 0.02% sodium azide), 100ml.
AB105-02 Preparation and wash Buffer for use when binding antigens to antibodies on the gel, (autoclaved PBS pH 7.4), 250ml.
AB108-01 Solution for pre-conditioning the Hydrazide Gel / ligand matrix to maximise antigen binding, (1M NaCl), 100ml.
AB103-02 Affinity buffer for eluting antigens from gel bound antibodies, (0.1M Glycine pH 2.5), 250ml.
AB107-01 Buffer for neutralising the pH 2.5 glycine buffer as it elutes from the column, (1M Tris HCl pH 8.0), 100ml.
AB501 Severn Biotech Fast Desalting Column Plus, (Stored in 0.05% sodium azide buffer). for removing sodium meta-periodate after oxodising glycoproteins and before adding them to the hydrazide column.

Severn-Link Hydrazide Agarose - Method Of Use

A) Attaching the glyco-protein or antibody to the agrose column.

Bring the Severn-Link Gel to room temperature, 20°C, before use. 

1. Push one of the frits supplied to the bottom of the 10 ml plastic column. Shake the bottle of Gel and carefully pour a 2 ml bed volume of Gel into the column. Do not allow the Gel bed to run dry at any stage. Wash the column with 12 ml of Preparation and Wash Buffer (AB105-02), and then place the end cap onto the base of the column.

2. Dissolve or dilute 1-10 mg glycoprotein in 1 ml Preparation and Wash Buffer (AB105-02).
(N.B If the protein is already dissolved in an unsuitable buffer, first change the buffer to the wash buffer, using the desalting column)

Add the protein solution to the amber vial containing 5 mg sodium meta-periodate (AB202-01). Swirl gently to dissolve the oxidising agent. Incubate the sample for 30 minutes at room temperature. The reaction is light sensitive and should be performed in the dark. As the oxidising agent is a salt desalting the ligand can stop the reaction. Using a Severn Biotech Fast Desalting Column Plus (AB501) desalt the now oxidised glycoprotein, (separate instructions enclosed).

3. Add the fraction containing the oxidised glycoprotein to the Severn-Link Gel column. Place the cap on top of the column and wrap both ends well with parafilm to prevent any leakages. 

4. React the Gel and protein with stirring or gentle agitation of the column for a minimum of 6 hours. You can use a roller method to do this in the tube.

5. Wash the column with 20mls of storage buffer and store at 4C.(do not freeze) the column is now ready for use.

B) For affinity purification of an Antigen or secondary antibody.

1. Wash the column again with 24ml of Preparation and Wash Buffer, (AB105-02). 
The column is now ready to affinity purify the antigen which must be in a solution of approximately neutral pH.

2. Pass the anti-serum or antigen solution through the column several times to ensure maximum binding of the antigen to the column.

A small peristaltic pump can be used to re-circulate the antigen solution through the column if desired. This ensures maximum binding of desired molecules.

3. The affinity-bound antigen can then be eluted from the column by adding 10 x 1ml aliquots of Elution Buffer, (AB103-02). Collect the antigen in 1ml fractions in tubes containing 100μl of Neutralising Buffer, (AB 107-01), and mix each fraction as it is collected.
Carry this step out quickly as possible so the antigen is not exposed to acidic conditions for longer than necessary.
The antigen will usually start to emerge in fraction 5 with a maximum quantity in fraction 7 after which all the antigen will have been eluted.

4. Immediately wash the column with 20mls storage buffer. This will ensure that the antibody on the column is exposed to acidic conditions for the minimum length of time. The column may then be re-useable although this would not always be so :it depends on the antibody , which differ in this respect.

For Further information on the use of Severn-Link Hydrazide Gel please contact technical sales
Severn Biotech Ltd. Unit 2 Park Lane,
Kidderminster
Worcestershire
DY116TJ
Tel:+44(0)1562-825286 Fax +44(0)1562-825284
Email info@severnbiotech.com