Affinity Chromatography

When red wine or tomato sauce stain a surface or fabric, they exhibit a well-known affinity Interaction. Through such everyday examples of affinity phenomena, we know that letting the stain dry makes it much harder - and sometimes impossible - to remove. The pH, ionic strength, stain concentration, temperature and residence time can all influence the outcome of any attempt to remove the stain.

With Affinity Interaction Chromatography (AIC) one is taking advantage of a very specific binding affinity between a ligand (L) and a target substance(T). Unlike older , more time-consuming chromatographic methods (SEC and IEC), agarose bead AIC can selectively separate ONE component from a complex mixture . This is done by passing the complex mixure over ligand-attached agarose beads, removing all substances not bound to the ligand and then breaking the L-T bond and eluting the desired T substance.

A Google search of "affinity chromatograpy" reveals a growing number of review articles on this subject. A search for a published AIC application -like the one you're contemplating- is recommended before beginning any AIC experiment . In general, however, these AIC review articles describe how the the ligand used can range from low molecular weight drugs, enzyme inhibitors or simple hydrorphobic moieties (as in HIC) to high molecular weight antibodies, enzymes and nucleic acids. Using the appropriate sized affinity beads( Re: bead diameter) , separations have been achieved over a wide range of target substance (T) sizes- ranging from low MW peptides or oligonucleotides to high MW proteins and DNA. Even intact cells and subcellular particles or inclusions can be separated using Cell Affinity Chromatography because the interbead channel can be made large enough to allow passage of intact cells - unless specifically bound by the ligand used.

The importance of bead porosity : Both the Ligand (L) and Target substance (T) must interact either within the pores of porous affinity beads or in the inter-bead channel. For this reason, the size of the pores is important in order to avoid unwanted steric hindrance of the L-T interaction within the porous matrix. Most of the AIC done with beads has been done with 4% or 6% agarose beads. That is, beads which have large macropores created by either a 4 % (w/v) or 6% (w/v) agarose gel composition. Such highly macroporous gels also allow for the maximum L- coupling and hence T-capture per unit volume of beads.

In recent years, one of the most exciting and rapidly growing uses of affinity techniques is Magnetic BioAffinity Separations(MBAS). With MBAS, one has far greater control over the ligand-target substance (L-T) residence time- because magnetic separations can be done much faster than their typical "drip-drip-drip" affinity column counterparts (see Magnetic Beads section for photo-illustrated methodology) . Using Magnetic Affinity Bioseparations (MBAS), the investigator can therefore avoid the L-T "aging" problem often associated with certain affinity ( incl. HIC) techniques. Such aging results in a tighter than desirable L-T binding which then causes either reduced levels of T recovery or requires the use of more extreme recovery conditions. Unfortunately, more extreme recovery procedures can also cause a reduction in the bioactivity of the recovered T- substance. Such limitations, of course, reduce the promise of AIC; but, with the advent of MBAS, such problems can be minimized through greater L-T residence time control.

Affinity Beads Selection Guide

Note: bead size is 50-150 µ for all beads in this section; also,
agarose concentration is either 4% (A-4B) or 6% (A- 6B) as indicated.

Pre-Activated Beads
Reference Brand Name BioScience Bead Equivalent (code) Settled Bead Volume Catalog Number BioScience Bead Price Target Application(s)
Sigma C9210; CNBr-Activated 4% Agarose A-4B-CL-OH*; Equivalent after CNBr or CDI activation; See "How To" Protocol Coupling to Ligand- NH2
$50/3.5 ml 100 ml A-0401-0100 $60.00
$171/17.5 ml 200 ml A-0401-0200 $96.00
$387/52.5 ml 400 ml A-0401-0400 $154.00
> 400 ml Inquire Inquire
Sigma C-9267; CNBr-Activated 6% Sepharose® A-6B-CL-OH*; Equivalent after CNBr or CDI activation; See "How To" Protocol Coupling to Ligand- NH2
$193/15 ml 100 ml A-0601-0100 $60.00
200 ml A-0601-0200 $100.00
400 A-0601-0400 $154.00
> 400 ml Inquire Inquire
Sigma P-9967 Periodate Activated Agarose A-4B-CL-OR; R = OCH2CHO; i.e. Glyoxal (G) 10 ml G-A-0401-0010 $30.00 Coupling to Ligand- NH2; requires NaCNBH3 reduction
$124/5ml 50 ml G-A-0401-0050 $216.00
100 ml G-A-0401-0100 $345.00
> 100 ml Inquire Inquire
Sigma P-3568 Periodate Activated Agarose; $82/5ml A-6B-CL-OR; R = OCH2CHO; i.e. Glyoxal (G) 10 ml G-A-0601-0010 $30.00 Coupling to Ligand- NH2; requires NaCNBH3 reduction
$164/10 ml  50 ml G-A-0601-0050 $216.00
$323/50 ml 100 ml G-A-0601-0100 $345.00
200 ml G-A-0601-0200 $552.00
> 200 ml Inquire Inquire
ABT Aminoethyl Agarose Bead A-4B-CL-OR; R = OCH2CH2NH2; Aminoethyl (AE) 10 ml AE-A-0401-0010 $58.00 Coupling to Ligand-CO2H; requires carbodiimide
$150/50 ml 50 ml AE-A-0401-0050 $140.00
$280/ 100 ml 100 ml AE-A-0401-0100 $250.00
$1050/500 ml 500 ml AE-A-0401-0500 $900.00
> 500 ml Inquire Inquire
ABT Aminoethyl Agarose Bead A-6B-CL-OR; R = OCH2CH2NH2; Aminoethyl (AE) 10 ml AE-A-0601-0010 $58.00 Coupling to Ligand-CO2H; requires carbodiimide
$150/50 ml 50 ml AE-A-0601-0050 $140.00
$280/ 100 ml 100 ml AE-A-0601-0100 $250.00
$1050/500 ml 500 ml AE-A-0601-0500 $900.00
> 400 ml Inquire Inquire
Sigma A-9019; CH-Sepharose®-4B A-4B-CL-OR; R = OCH2CO2H ; Carboxymethyl (CM) 50 ml CM-A-0401-0050 $46.00 Coupling to Ligand- NH2; requires carbodiimide
$265/15 ml 100 ml CM-A-0401-0100 $75.00
$595/45 ml 200 ml CM-A-0401-0200 $120.00
400 CM-A-0401-0400 $210.00
> 400 ml Inquire Inquire
Sigma CCl-6B-100-CL A-6B-CL-OR; R = OCH2CO2H ; Carboxymethyl (CM) 50 ml CM-A-0601-0050 $46.00 Coupling to Ligand- NH2; requires carbodiimide
$67/50 ml 100 ml CM-A-0601-0100 $75.00
$112/100 ml 200 ml CM-A-0601-0200 $120.00
$353/500 ml 400 CM-A-0601-0400 $210.00
> 400 ml Inquire Inquire
Protein- Binding Ligands
Reference Brand Name BioScience Bead Equivalent (code) Settled Bead Volume Catalog Number BioScience Bead Price Target Application(s)
Sigma A-1018; Arg-coupled 6 % Agarose Bead Arginine-coupled, 4% agarose; A-CL-O-R ; R= CH2CH2-Arg 5 ml Arg-G-A-0401-0005 $100.00 Arginine binding enzymes and proteins
$157/5ml 25 ml Arg-G-A-0401-0025 $300.00
$522/25ml > 25ml Inquire Inquire
Sigma A-1018; Arg-coupled 6 % Agarose Bead Arginine-coupled, 6% agarose; A-CL-O-R ; R= CH2CH2-Arg 5 ml Arg-G-A-0601-0005 $110.00 Arginine binding enzymes and proteins
$157/5ml 25 ml Arg-G-A-0601-0025 $365.00
$522/25ml > 25 ml Inquire Inquire
Closest analog: Sigma S-1799; SP-Sepharose® C-4B-CL-OSO3Na ; C= carrageenan ; (NaCgn) 10 ml C-0401-0010 $40.00 Heparinoid, Reverse transcriptase, and virus binding
50 ml C-0401-0050 $70.00
$154/100 ml 100 ml C-0401-0100 $108.00
$518/500 ml 500 C-0401-0500 $363.00
> 500 ml Inquire Inquire
Sigma H-6508; Heparin-Agarose Heparin-coupled, 4% agarose; A-CL-O-R ; R= DVS-heparin Heparin-binding proteins and enzymes
$51/ 5ml 5 ml Hep-A-0401-0005 $40.00
$176/25 ml 25 ml Hep-A-0401-0025 $110.00
$533/100ml 100 ml Hep-A-0401-0100 $330.00
> 100 ml Inquire Inquire
Sigma M-6400; D-Mannose- coupled, CL, 4% agarose bead; Mannose-coupled, 4% agarose; A-CL-O-R ; R= DVS-mannose Binds > 50 mg/ml of Concanavilin-A ( Con-A)
$131/5ml 5 ml MO-A-0401-0005 $90.00
$232/10ml 10 ml MO-A-0401-0010 $144.00
$510/25 ml 25 ml MO-A-0401-0025 $288.00
> 25 ml Inquire Inquire
Sigma M-9917; Mannan- coupled, CL, 4% agarose bead; Mannan-coupled, 4% agarose; A-CL-O-R ; R= DVS-mannan Binds > 30 mg/ml of Concanavilin-A ( Con-A)
$102/5ml 5 ml MA-A-0401-0005 $72.00
10 ml MA-A-0401-0010 $115.00
$399/25 ml 25 ml MA-A-0401-0025 $230.00
50 ml MA-A-0401-0050 $368.00
100 ml MA-A-0401-0100 $588.00
>100 ml Inquire Inquire
Carbohydrate-Binding Ligands
Reference Brand Name BioScience Bead Equivalent (code) Settled Bead Volume Catalog Number BioScience Bead Price Target Application(s)
Sigma # 18,4454 resin bead Boric acid-coupled-4% agarose; A-CL-O-BO2-R ; R= polymannan Binds mannose
$74/5ml 5 ml BO-A-0401-0005 $28.00
10 ml BO-A-0401-0010 $40.00
$302/30 ml 25 ml BO-A-0401-0025 $80.00
50 ml BO-A-0401-0050 $128.00
100 ml BO-A-0401-0100 $205.00
>100 ml Inquire Inquire
Other Specific Ligands
Reference Brand Name BioScience Bead Equivalent (code) Settled Bead Volume Catalog Number BioScience Bead Price Target Application(s)
Various Available on request Inquire Inquire Inquire Inquire

A=Agarose; 4 or 6 = % Agarose in gel bead; CL=crosslinked; R= ether-linked substituent or derivative; DVS= divinyl sulfone.

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