Ovalbumin chromatography

Loading capacities in size exclusion chromatography are very low because all separation occurs within the liquid volume of the column. The small diffusion coefficients of macromolecules also contribute to bandspreading when loads are increased. The mass loading capacities for ovalbumin (MW 45,000) on various sizes of columns can be seen in Table 10.5. The maximum volume that can be injected in size exclusion chromatography before bandspreading occurs is about 2% of the liquid column volume. The maximum injection volumes for columns of different dimensions can also be seen in Table 10.5. 

Figure 9.6 Surfer-generated chromatoeletropherogram of fluorescamine-labeled tryptic digest of ovalbumin. Reprinted from Analytical Chemistry, 62, M. M. Bushey and J. W. Jorgenson, Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography/capillary zone electrophoresis, pp 978-984, copyright 1990, with permission from the American Chemical Society.

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Organotin compounds la 399 lb 21, 319 Ornithine la 235 Orthosipon leaf extract lb 216,217 Oryzalin lb 110-112 Ovalbumin lb 401 Over pressure layer chromatography (OPLC) lb4 Oxacillin lb 84,188,301 Oxaflozan lb 268,358 Oxalic acid la 45,171,426 Oxamyl lb 332... 

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

New designs for axial flow process chromatography columns have been examined using ovalbumin separation on Whatman Express-Ion Exchanger Q with a 16 L Side-Pack and a 24 L IsoPak column.38 The Side-Pak column is packed in the transverse direction, so radial inhomogeneity is minimized. 

FIGURE 1.3 Three-dimensional representation of a tryptic digest of ovalbumin. The three-dimensional separation consists of size-exclusion chromatography (first dimension), reversed-phase LC (second dimension), and capillary electrophoresis (third dimension). From Moore and Jorgenson, (1995) with permission of the American Chemical Society. 

Figure 4.9 Anion-exchange liquid chromatography of acidic proteins. Column, Asahipak ES502N eluent, 20 min linear gradient of sodium chloride from 0 to 500 pM in 50 mM bis-tris/HCl buffer pH 7.0 flow rate, 1ml min-1 detection, UV 280 nm. Peaks-. 1, conalbumin (Mr 77000-88000, pi 6.0-6.8) 2, ovalbumin (Mt 45000, pi 4.6) 3, trypsin inhibitor (Mr 8000, pi 4.5) and 4, / -lactoglobulin (Mr 18400, pi 5.1).

Figure 6. Aqueous size exclusion chromatography of the APPL derived from S. badius. Ferritin, aldolase, ovalbumin, chymotrypsinogen A and acetone were used as standards.

Yamashina and coworkers18 72 isolated, on Dowex 50, the total aspartamidoglycan from a proteolytic digest of ovalbumin. It had mean D-mannose hexosamine L-asparagine ratios of 5 3 1. Purified a-D-mannosidase liberated exactly half the D-mannose from the glycopeptide. The residue was subjected to chromatography on Bio-Gel... 

These fractions, altogether, account for only 30% of the mannose in the ovalbumin digest before separation by ion-exchange chromatography. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Figure 2.9 Hydrophobic-interaction chromatography of proteins. (A) Ammonium sulfate gradient from 2.16 to 0 M (B) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 10 mM, respectively (C) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 20 mM, respectively (D) ammonium sulfate and tetrabutylammonium bromide gradients from 2.16 to 0 M and from 0 to 40 mM, respectively. Chromatography conditions column, silica-bound polyether, 10 cm x 4.6 mm I.D. temperature, 25°C flow rate, 1 ml/min gradient, linear for 30 min background buffer, 50 mM phosphate, pH 6.5. Peaks a, cytochrome c b, ribonuclease A c, /3-lactoglobulin A d, lysozyme e, ovalbumin f, a-chymotrypsinogen A g, fetuin. (Reprinted from Ref. 45 with permission.)...

Figure 2.12 Protein separation by anion-exchange chromatography. Chromatography conditions column, Hydrophase HP-SAX eluent A, 5 mM Bis-Tris, pH 8.35 eluent B, A plus 0.5 M NaCl gradient, 0-50% B over the times indicated temperature, ambient detection, UV absorbance at 280 nm injection, 100 ml. Peaks 1, myoglobin 2, cytochrome c 3, ovalbumin. (Reprinted from Ref. 51 with permission.)...

Figure 1. SDS-PAGG analysis of alkaline-dissolved Bacillus thuringiensis subspecies kurstaki (BTK) and israelensis (BTI) -endotoxin at 25 yg per track (1) BTK -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.), (2) BTI 6-endotoxin from Sandoz Inc., (3) BTI (Sandoz) -endotoxin purified by DEAE-anion exchange chromatography, (4) percipitate formed after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (5) soluble fraction after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (6) BTI (Sandoz) -endotoxin purified by Sephadex G-75 gel filtration chromatography at Rf 1.35, (7) at Rf 1.58, and (8) BTI strain IFC-1 -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.). S, molecular weights as indicated X1000 for bovine serum albumin (BSA), ovalbumin (OA), trypsin, and myoglobin. Reproduced with permission from Ref. 29. Copyright 1984, Academic Press, Inc.

Figure 5. SDS-Page analysis of alkaline-dissolved Bacillus thuringiensis israelensis (BTI) 6-endotoxin from Sandoz Inc. at 25 yg per track (1) BTI 6-endotoxin as prepared in Figure 1 (Track 2), (2) soluble fraction after dialysis of BTI 6-endotoxin into pH 4.5 sodium acetate buffer, and (3) 25K component from BTI 6-endotoxin after pH 4.5 percipitation and DEAE-anion exchange chromatography. S, molecular weight markers from top to bottom bovine serum albumin (68K daltons), ovalbumin (43K), and myoglobin (16K).

Fig 2. Rapid reversed-phase chromatography of standard proteins. Supports RP-300 (panels A B) Poros RII/H (panels C D). Chromatographic conditions linear 6-ml gradient of 0-100%B. Solvent A aqueous 0.1% TFA, Solvent B aqueous 0.1% TFA containing 60 % acetonitrile. Temperature 45°C. Chromatographic runs performed at superficial linear flow velocities of 173 cm/h (0.1 ml/min) (A, C) and 3465 cm/h (2.0 ml/min) (B, D). Proteins (5 pg) 1, ribonuclease-B 2, chick lysozyme 3, bovine serum albumin 4, myoglobin 5, carbonic anhydrase 6, ovalbumin. 

Ovalbumin, 5%, 0.25 ml in 0.15 M phosphate, pH 7.1 Procedure. To the Na H add the PA, followed by chloramine-T. Shake the reaction mixture for 1 min at 23°, then add sodium metabisulfite followed by ovalbumin. [ H]PA is isolated by chromatography on a column (1.5 X 30 cm) of Sephadex G-25. Although the authors suggest that the product be stored at - 20°, precipitation and inactivation can occur under these conditions. Storage at 4° in 0.04% sodium azide is recommended. ... 

Fig. 4 The total ion chromatography (TIC) of the separation of a tryptic digest of chicken ovalbumin with a sample injection amount of 12 pmol corresponding to the original protein [52]. Column length, 6 cm. Conditions 20 min, 0-40% acetonitrile gradient 1000 V applied voltage with a 40-bar supplementary pressure. (From Ref. 52 reproduced with permission of the authors and the American Chemical Society.)...

Recently, PHEMA microspheres have been more and more extensively used for IMAC. Separon-IDA-Fe(III) or Cu(II) was prepared. It was found that Cu(II) interacted preferentially with histidine and tryptophan residues, while Fe(III) preferred phosphate residues, as demonstrated by the separation of lysozyme, ribonuclease A, myoglobin, and transferrin on the Cu(II) column and ovalbumin on the Ee(III) column, respectively.The PHEMA-Congo Red-Cu(II) and PHEMA-Cibacron Blue E3GA-Zn(II) microspheres were applied for adsorption of BSA. Without incorporating the metal ions, the dyed sorbents were already good stationary phases for affinity chromatography. As shown in Fig. 2, the addi-... 

---