Bio-rad Affi-Gel 15 Gel User Manual

Activated ImmunoaffinitySupportsCatalog Numbers153-6046 Affi-Gel®10 Gel153-6052 Affi-Gel 15 Gel153-6098 Affi-Gel 10 and 15 GelLIT156B 6/17/98 10:40 A

Fig. 4. Effect of amount of protein added on protein cou-pling to Affi-Gel 10 gel. A similar coupling capacity isobserved with Affi-Gel 15 gel.mg prot

3. Closed container which holds at least four times thevolume of gel.4. (Optional) rotating shaker.5. (Optional) Buchner funnel.Shake the vial, and ma

5.2 Anhydrous CouplingCoupling under anhydrous condition is the preferredmethod when the solubility of the ligand permits. It isideal for peptides. Si

Any unreacted groups that remain can be blocked byaddition of a slight excess of ethanolamine at the end ofthe reaction. The resulting support will ha

• Affi-Gel support is more than 12 months old. Try newmaterial.• The Affi-Gel support has been stored too warm. • pH is not optimal. For Affi-Gel 10 g

Section 8Using the Coupled SupportWhen the Affi-Gel 10 or 15 activated support hasbeen coupled to the ligand, it is ready to use. General operating co

Optimizing Support and Sample VolumesUse only the required amount of affinity support. Ifexcess support is used, sample elution becomes more dif-ficul

Another method is to titrate the gel with sample,checking the supernatant for unbound sample after eachaddition. This can be done either in a column o

1. Specific Elution with excess antigen or antibodyshould be considered first, because, in theory, it willalways work. It is often impractical due to

hydrophobic interactions. Chaotropic anions areeffective in the order SCN->ClO-4>I>Br->Cl-.6,7Chaotropic cations are effective in the orde

Table of ContentsSection 1 Introduction... 1Section 2 Coupling Chemistry... 2Section 3 Genera

Alternative procedures have been published for elut-ing labile antigens from immobilized antibody columns.Deionized water has been reported,8,9but yie

Catalog Number Product Description CommentsDesalting and sample preparation150-0738 Bio-Gel P-6DG Desalting Rapid protein and peptide Gel, 100 g desa

Section 11References1. Prickett, K. S., et al., BioTechniques, 7, 580 (1989).2. Frost, R., et al., Biochem. Biophys. Acta., 670, 163 (1981).3. Izuta,

Section 1IntroductionAffi-Gel 10 and Affi-Gel 15 affinity supports are acti-vated immunoaffinity supports that offer rapid, high effi-ciency coupling

tive ester immobilized on the gel is highly selective forprimary amino groups, spurious side reactions with theligand (i.e., cross-linking or other mo

Section 3General Coupling Conditions3.1 pH DependenceA major advantage of Affi-Gel 10 and 15 supports isthe mild conditions which will permit coupling

will repel negatively charged proteins (proteins bufferedat a pH above their isoelectric point) and lower their cou-pling efficiency. The Affi-Gel 15

Table 1. Protein Coupling to Affi-Gel 10 andAffi-Gel 15 SupportCoupling Efficiency (%)Protein pl Affi-Gel 15 Gel Affi-Gel 10 Gel1. Fetuin 3.3 43 3.02.

Table 2. Coupling Efficiency of Acidic andNeutral-to-Slightly-Basic Protein Under VariousCoupling ConditionsAffi-Gel 10 Affi-Gel 15Coupling CouplingCo

protein coupled per ml of gel (Figure 4). The efficiency ofcoupling will vary with the protein and conditions of cou-pling (Figure 1). Greater than 30