Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses, and cells (bacteria): Ia. gel antibodies against proteins (transferrins)

Artificial antibodies in the form of gel granules were prepared by the molecular imprinting technique from the monomers acrylamide and N,N'-methylene-bis-acrylamide. Gel granules, freed from the selectively adsorbed protein (the antigen), are neutral and, accordingly, do not migrate in an electrical field. However, upon selective interaction with the antigen at a pH different from its pI, the granules become charged. The selectivity of the gel antibodies was studied by free zone electrophoresis in a tube with inside diameter larger than the size of the granules. Such electrophoretic analyses showed that gel antibodies against iron-free transferrin had a high selectivity for this protein, although some crossreaction took place with iron-saturated transferrin, indicating that these artificial antibodies can easily distinguish the minute differences in the 3-D structure of the transferrins. Analogously, gel antibodies against iron-saturated transferrin were highly selective for this protein with some crossreaction with iron-free transferrin. The mobilities of iron-free and iron-saturated transferrin are very similar, and, therefore, capillary free zone electrophoresis cannot distinguish between these structurally related proteins. However, significant differences in the mobilities of the selective gel granules can be observed depending on their interaction with iron-free or iron-saturated transferrin, i.e., the artificial gel antibodies may become powerful analytical tools.     

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.     

Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density and salt concentration on the retention of peptides

The adsorption characteristics of a variety of synthetic peptide hormones and di-, tri- and tetrapeptides on Cu(II) immobilized on two commercially available high-performance chelating gels run under various experimental conditions are described. Methods for determining the concentration of immobilized Cu(II) in situ are also described. The Cu(II)-charged columns exhibit a net negative charge as judged from the significantly higher retention of some basic peptides in the absence of NaCl in the equilibration and elution buffers. At higher NaCl concentrations (2-4 M), aromatic interactions seem to be superimposed on the metal ion affinity characteristics of the peptides. The relationship between resolution of peptides and the concentration of immobilized Cu(II) ions has also been established for the Chelating Superose gel where 40 mumol Cu(II) ml-1 gel apparently gives the optimum resolution. The nature of the gel matrix also plays a role in the resolution of some peptides, the extent of which is difficult to predict. The results obtained also suggest that peptides containing aromatic and hydroxy amino acids are retarded more than those which lack them. Moreover, these same amino acids apparently strengthen the existing strong binding of peptides containing His, Trp or Cys to a Chelating Superose-Cu(II) column. Dipeptides with C-terminal His (i.e., X-His) are neither bound nor retarded on a column of Chelating Superose-Cu(II) whereas those having the structure His-X are strongly bound. Some tri- and tetrapeptides containing His were also found not to bind to the column. The underlying cause of this anomalous adsorption behaviour is discussed and is ascribed to "metal ion transfer" arising from the relatively higher affinity of such peptides towards immobilized Cu(II) ions than the chelator groups (iminodiacetate) which are covalently bound to the gel matrix.     

Nickel and copper complexes of a chelating methacrylate sorbent in the purification of chitinases and specific immunoglobulin G1 by immobilized metal ion affinity chromatography

The isolation of the isoforms of endo- and exochitinases of Clostridium aminovalericum T1 and of the horseradish peroxidase (HRP)-specific immunoglobulin G1 from natural sources by immobilized metal ion affinity chromatography was studied. The effect of Cu2+ and Ni2+ complexes of iminodiacetic acid incorporated in porous glycidyl methacrylate-co-ethylene dimethacrylate and in agarose (Sepharose Fast Flow) beads on separation of the target polypeptides was analyzed. It was found that the Cu2+ complexes bound both the HRP-specific IgG1 and some isoforms of chitinases more strongly than the Ni2+ complexes. From the former complexes, both target polypeptides were eluted by a stepwise imidazole concentration gradient of 5-100 mM. The lower strength of Ni2+ complex binding with the HRP-specific IgG1 resulted in its easy elution with a pH gradient of 5.5-5 while some isoforms of chitinases required imidazole for their elution. The "fraction elution degree" of a target polypeptide (i.e., the ratio of its amounts in each eluate fraction and in the combined fractions) was used for the evaluation of the sorption selectivity and binding affinity of the separating components to the studied metal complexes.     

IMAC—Immobilized metal ion affinity based chromatography

Immobilized metal ion affinity chromatography (IMAC) is a highly versatile separation method based on interfacial interactions between biopolymers in solution and metal ions fixed to a solid support, which is usually a hydrophilic cross-linked polymer.Polymer-fixed Zn(II), Ni(II), Co(II) and Cu(II) are particularly well suited for fractionation of proteins primarily on the basis of their relative content of surface-located imidazole residues but also of Trp and Cys residues as well as terminal amino groups.IMA methods can also be devised for purification of phosphoproteins and calcium-binding proteins. In some instances, the performance of IMA gels is comparable to that of biospecific affinity-based adsorbents. In fact IMA gels may, by sandwich techniques, occasionally be converted to biospecific adsorbents.Selectivity can be varied by choice of type of ligand and metal ion as well as by varying the modes of elution, including affinity desorption.     

pH titration curves of the desialylated human alpha 1-acid glycoprotein variants by combined isoelectrofocusing-electrophoresis: utilization in the development of a fractionation method for the protein variants by chromatography on immobilized metal affinity adsorbent.

Using a two-dimensional isoelectrofocusing (IEF)-electrophoresis technique, the pH titration curves of the three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG) were studied to assist in the development of a fractionation method for the AAG variants. For this purpose, different AAG samples, each corresponding to one of the three main phenotypes of the protein (F1S/A, F1/A and S/A), were first purified by chromatographic separation of individual human plasma samples on immobilized Cibacron Blue F3G-A. The purified AAG samples were then disialylated and their heterogeneity was checked by analytical IEF. The pH-mobility curves of the desialylated AAG samples were displayed in polyacrylamide gel slabs, under a constant set of experimental conditions, by carrying out electrophoresis of the protein samples perpendicularly to two stationary pH gradients: a large gradient (pH 3.5-9.5) and a narrow gradient (pH 5-8). The curves showed that all the desialylated variants of AAG exhibited small charge differences and moved closely together between about pH 3.5-5.5 and pH 7.5-9.5. However, the variants were found to show microheterogeneity in their total charge between about pH 5.5 and 7.5 due to the titrated ionizable groups involved along this pH zone. This microheterogeneity was assumed to be accounted for by the existence of differences between the titratable histidyl residues of the AAG variants. Consequently, the interactions of the variants with immobilized transition metal ions were studied at pH 7, using affinity chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the A variant was strongly bound by immobilized Cu(II) ions, whereas the F1 and S variants interacted non-specifically with the immobilized ligand. This finding allowed the development of a rapid and effective fractionation method for desialylated AAG into its A and F1 or S variants, depending on the AAG phenotype, by chromatography on an immobilized affinity Cu(II) adsorbent. The quantitative relationships between immobilized Cu(II) ions and desialylated AAG (the apparent association constant and gel protein-binding capacity) were also determined using a non-chromatographic equilibrium binding technique.     

Presence of imidazole in loading buffer prevents formation of free radical in immobilized metal affinity chromatography and dramatically improves the recovery of herpes simplex virus type 1 gene therapy vectors

We have recently shown that immobilized metal affinity chromatography (IMAC) is an effective technique for purification of herpes simplex virus type 1 (HSV-1) gene vector engineered to display cobalt affinity tag on the envelope. However, the tagged HSV-1 viruses were severely inactivated by oxidative hydroxyl free radicals when crude HSV-1 supernatant was applied on an immobilized cobalt column and eluted by a low pH buffer. Furthermore, we have reported that virus inactivation could be prevented by inclusion of high concentration of ascorbate in chromatographic mobile phase. In this paper we report that when elution of bound virus was attempted by inclusion of imidazole in elution buffer, rather than lowering the pH of elution buffer, similar inactivation was also observed. The results also demonstrated that virus inactivation was dramatically reduced by inclusion of 20mM imidazole in the loading buffer. Electron spin resonance (ESR) experiments suggest that imidazole prevents hydroxyl free radical generation from the cobalt complexes. This is the first report describing the role of imidazole in preventing free radical formation in an IMAC column. From a practical stand point, our results imply that inclusion of appropriate amount of imidazole in the loading buffer is an effective strategy for improving the recovery yield of active products and for enhancing product quality during IMAC purification.     

固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶

以Ｃｕ~（２＋）－Ｓｅｐｈａｒｏｓｅ４Ｂ固定化金属离子亲和色谱法纯化猪铜锌超氧化物歧化酶,考察了不同的洗脱缓冲溶液及其ｐＨ对纯化效率的影响,显示了方法的有效性。     

Interactions of Ni(II) and Cu(II) ions with the hydrolysis products of the C-terminal -ESHH- motif of histone H2A model peptides. Association of the stability of the complexes formed with the cleavage of the -E-S- bond

We studied the interactions of Ni(II) and Cu(II) ions with the synthetic tetrapeptides SHHK- and SAHK-, which were blocked by amidation making them more realistic models of the hydrolysis peptidic products of the hexapeptides models of H2A histone. A combination of potentiometric and spectroscopic techniques (UV/Vis, CD, NMR and EPR) suggested that at pH > 7 both tetrapeptides coordinated equatorially through the imidazole ring of His in position 3, the N-terminal amino group and the two amide nitrogens existing between these groups {NH2, 2N-, NIm} forming 4N square-planar complexes. While in the case of the CuH(-1)L complex with SHHK- a possible axial coordination of the imidazole ring of His in position 2 was suggested, in the case of the analogous NiH(-1)L complex a completely different interaction of the same ring with metal ions was observed. As expected these complexes have the same structures with the hydrolysis products produced from the Ni(II)- or Cu(II)-assisted hydrolysis of previously studied hexapeptide models of the C-terminal of histone H2A, due to their predominance at pH > 7.4. In addition, the competition plots presented herein showed that the synthetic tetrapeptides SHHK- and SAHK- have higher affinity towards Ni(II) and Cu(II) ions than the previously studied hexapeptides, suggesting that metal ions remain bound to the peptidic products during the hydrolysis cleavage. Thus, it can be concluded that the stability of Ni(II) or Cu(II) complexes with the synthetic tetrapeptides and consequently with the real hydrolysis peptidic products is the driving force of the hydrolysis reaction of H2A histone blocked hexapeptide models, presented in previous studies.     

蛋白折叠液相色谱法复性和纯化大肠杆菌表达的重组人粒细胞集落刺激因子

用蛋白折叠液相色谱法(PFLC)对大肠杆菌表达的包涵体形式的重组人粒细胞集落刺激因子(rhG-CSF)进行了复性并同时纯化。用Cu2+-亚氨基二乙酸（IDA） Sepharose作为固定化金属离子亲和色谱的固定相。在低浓度脲存在下,以咪唑为洗脱剂,采用线性梯度洗脱rhG-CSF。该法仅通过一步PFLC分离,减少了复性过程中rhG-CSF的聚集,复性后的rhG-CSF的比活性为1.8×108 IU/mg,纯度为97%,质量回收率为32%。     

Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. II. Immobilization of synthetic metal-binding peptides from metal ion transport proteins as model bioactive protein surface domains.

This preliminary investigation tests the premise that biologically relevant (1) peptide-metal ion interactions, and (2) metal ion-dependent macromolecular recognition events (e.g., peptide-peptide interactions) may be modeled by biomimetic affinity chromatography. Divinylsulfone-activated agarose (6%) was used to immobilize three different synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein histidine-rich glycoprotein (HRG). The synthetic peptides represented 1-3 multiple repeat units of the 5-residue sequence (Gly-His-His-Pro-His) found in the C-terminal of HRG. By frontal analyses, immobilized HRG peptides of the type (GHHPH)nG, where n = 1-3, were each found to have a similar binding capacity for both Cu(II) ions and Zn(II) ions (31-38 mumol/ml gel). The metal ion-dependent interaction of a variety of model peptides with each of the immobilized HRG peptide affinity columns demonstrated differences in selectivity despite the similar internal sequence homology and metal ion binding capacity. The immobilized 11-residue HRG peptide was loaded with Cu(II) ions and used to demonstrate selective adsorption and isolation of proteins from human plasma. These results suggest that immobilized metal-binding peptides selected from known solvent-exposed protein surface metal-binding domains may be useful model systems to evaluate the specificity of biologically relevant metal ion-dependent interaction and transfer events in vitro.     

On the nature of immobilized tris(carboxymethyl)ethylenediamine

Immobilized tris(carboxymethyl)ethylenediamine (TED) (also known as ethylenediamine-N,N,N'-triacetic acid) serves as a stationary phase for fractionation of proteins and the preparation of metal-free proteins by immobilized metal-ion affinity chromatography. The Cu(II) complex of commercially available immobilized TED has been characterized by elemental, electrochemical and spectroscopic techniques. There is a large discrepancy between the theoretical capacity determined from the nitrogen content and the experimental capacities determined by atomic-absorption spectroscopy and anodic stripping voltammetry (ASV), indicating that a substantial portion of the immobilized ligand is not binding Cu(II). In addition, the titration of immobilized TED with Cu(II), monitored by ASV, suggests that more than one ligand is involved in binding Cu(II). A comparison of the EPR spectrum for immobilized Cu(II)-TED with spectra for various model complexes shows that the major immobilized ligand is ethylenediamine-N,N'-diacetic acid (EDDA) with immobilized TED present only as a minor component. The complexation constant for Cu(II) is close to the value for Cu(II)-EDDA in solution. The formation of EDDA is consistent with the method for synthesizing immobilized TED.     

Examination of the binding behaviour of several proteins with the immobilized copper(II) complexes of o-, m- and p-xylylene bridged bis(1,4,7-triazacyclononane) macrocycles

Three new IMAC chelating systems, incorporating immobilised xylenyl-bridged bis(1,4,7-triaza-cyclonane) ligands, complexed with Cu(2+) ions to form binuclear species, have been prepared. Their binding properties have been investigated with three small globular proteins (hen egg white lysozyme, horse skeletal muscle myoglobin and horse heart cytochrome c). The effects of buffer pH, ionic strength and composition on the binding behaviour of these proteins to these new IMAC sorbents have been examined and compared with those found for the corresponding immobilized mononuclear copper complex of 1,4,7-triazacyclononane (tacn). Higher protein binding affinities were observed with the Cu(2+)-bis(tacn) sorbents compared to the Cu(2+)-tacn system, consistent with the immobilized binuclear copper(II) species undergoing enhanced coordinative interaction with the surface-exposed histidine residues of these proteins. Moreover, the protein binding characteristics of these IMAC sorbents at higher ionic strengths, such as 1M NaCl, also reflect the presence of the aromatic ring in the bis(tacn) ligands, whereby hydrophobic pi/pi stacking interactions can occur with the proteins.     

Structure,insertion electrochemistry,and magnetic properties of a new type of substitutional solid solutions of copper,nickel, and iron hexacyanoferrates/hexacyanocobaltates

Substitutional solid solutions of metal hexacyanometalates in which low-spin iron(III) and cobalt(III) ions populate the carbon-coordinated sites were synthesized and studied by powder diffraction including Rietveld refinement, cyclic voltammetry of immobilized microparticles, diffuse reflection vis-spectrometry, and magnetization techniques. The continuous solid solution series of potassium copper(II), potassium nickel(II), and iron(III) [(hexacyanoferrate(III))(1-x)(hexacyanocobaltate(III))(x)] show that the substitution of low-spin iron(III) by cobalt(III) in the hexacyanometalate units more strongly affects the formal potentials of the nitrogen-coordinated copper(II) and high-spin iron(III) ions than those of the remaining low-spin iron(III) ions. In the case of copper(II) and iron(III) [(hexacyanoferrate(III))(1-x)(hexacyanocobaltate(III))(x)] the peak currents decrease much more than can be explained by stoichiometry, indicating that the charge propagation is slowed by the substitution of low-spin iron(III) by cobalt(III). The Rietveld refinement of all compounds confirmed the structure initially proposed by Keggin for Prussian blue and contradicts the structure described later by Ludi. The dependencies of lattice parameters on composition exhibit in all series of solid solutions studied similar, although small, deviations from ideality, which correlate with the electrochemical behavior. Finally, a series of solid solutions of the composition KNi(0.5)(II)Cu(0.5)(II)[Fe(III)(CN)(6)](1-x)[Co(III)(CN)(6)](x), where both the nitrogen- and carbon-coordinated metal ions are mixed populated and were synthesized and characterized. These are the first examples of solid solutions of metal hexacyanometalates with four different metal ions, where both the nitrogen- and the carbon-coordinated sites possess a mixed population.     

高效亲和色谱法测定丹皮酚与固定化人血清白蛋白结合域

利用亲和色谱,在模拟人体生理环境下(37 ℃、pH 7.4),采用竞争置换法研究了丹皮酚(PAE)与固定化人血清白蛋白(HSA)的相互作用。通过对PAE的自我竞争分析及PAE与HSA上结合位点的标记物间的竞争置换分析,得到了PAE和HSA间的结合常数、结合位点数和结合域。结果表明: PAE在HSA分子中仅存在一类结合位点,结合常数为4.84×103 L/mol,该结合位点为HSA上的Sudlow siteII;通过对PAE与HSA相互作用的热力学研究,推断出二者间的作用力类型为氢键或范德华力。     

Imidazole—a new ligand for metal affinity precipitation

Kunitz soybean trypsin inhibitor (STI) was specifically coprecipitated during precipitation of Cu(II)-loaded copolymers induced by increase in temperature and ionic strength. The copolymers used consisted of 1-vinylimidazole andN-vinylcaprolactam orN- isopropylacrylamide. The elution of STI was achieved by solubilization of the STI-Cu(II)-polymer complex in the presence of an excess of the competing ligand, imidazole, and a subsequent precipitation of the polymer with STI remaining free in solution in a purified form as judged by Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To the best of our knowledge this is the first reported successful metal affinity precipitation of protein in a heterobifunctional format.     

Adsorption of Cu(II) and Ni(II) on solid humic acid from the Azraq area, Jordan

Isotherms of adsorption of Cu(II) and Ni(II) onto solid Azraq humic acid (AZHA) were studied at different pH (2.0-3.7) values and 0.1 M NaClO4 ionic strength. The Langmuir monolayer adsorption capacity was found to range from 0.1 to 1.0 mmol metal ion/g AZHA, where Cu(II) has higher adsorptivity than Ni(II). The previously reported NICA-Donnan parameters for sorption of Cu(II) on HA fit the amount of Cu(bound) determined in the present study at pH 3.7 but underestimates those at pH values of 3.0, 2.4, and 2.0. The contribution of low affinity sites to binding of metal ions increases with decreasing pH and increasing metal ion loading. The aggregation of HA, which is facilitated by decreasing pH and increasing metal loading, may increase the ability of low-affinity sites to encapsulate metal ions. The binding of Ni(II) to HA exhibits less heterogeneity and less multidentism than that of Cu(II). AZHA loaded with Cu(II) and Ni(II) was found to be insoluble in water with no measurable amount of desorbed metal ions.     

Simultaneous Solid Phase Chelate Extraction for Ultratrace Determination of Copper,Nickel, and Zinc by Microsample Injection System Coupled Flame Atomic Absorption Spectrometry

A simple and accurate solid phase extraction method was developed for the determination of Cu(II), Ni(II), and Zn(II) at trace levels by microsample injection system coupled flame atomic absorption spectrometry. The proposed method is based on the retention of complexes of metal ions with 4-(2-pyridylazo) resorcinol on Amberlite XAD-7 resin and elution with acetone. The possible parameters influencing the preconcentration were optimized. The interferences of coexisting ions were studied. Under the optimal conditions, the detection limits of Cu(II), Ni(II), and Zn(II) were 0.058, 0.013, and 0.027 µg L^?1, respectively. The applicability and the accuracy of the proposed method were validated by analysis of BCR 715 wastewater as a certified reference material and spiked real samples. Recoveries in the range of 94–105% were obtained with relative errors less than 7.5% and relative standard deviations less than 3.2%.     

High-performance metal chelate affinity chromatography of cytochromes P-450 using Chelating Superose.

High-performance metal chelate affinity chromatography [immobilized metal ion affinity chromatography (IMAC)] using Chelating Superose (iminodiacetic acid adsorbent) was investigated for its suitability in purifying phenobarbital-induced rat liver microsomal cytochrome P-450 isozymes (P450) and optimized for preparative purposes. Starting with an 8-aminooctyl-Sepharose fraction of partially purified P450, it was found that only Ni(2+)- and Cu(2+)-charged columns could bind P450. No binding was ever observed when Zn2+, Co2+, Mn2+, Cd2+, Fe3+, Fe2+ or Tl3+ ions were employed. Of eight commonly used elution buffers, imidazole and tryptamine were found to cause some denaturation of P450. For desorption of proteins bound to Ni(2+)-charged columns, the following order of decreasing elution buffer strength was determined: cysteine approximately histidine greater than glycine greater than histamine greater than tryptophan greater than ammonium chloride. During protein desorption with some of these buffers, metal ions were found to bleed from the gel, resulting in P450 denaturation. This could be eliminated by prebleeding the charged columns prior to sample application and had an effect on product recovery and homogeneity. Ni2+ and glycine were chosen as a standard for further optimization involving sample adsorption conditions as influenced by equilibration buffer, detergent, load capacity and flow, gradient and temperature conditions. In this way, potassium phosphate (pH 7.75) and 0.4% Emulgen 911 were used to equilibrate a 1.6-ml column and purify 20-50 nmol of P450 (5-15 mg of protein) within 15 min. One gradient fraction consisted of a single sodium dodecyl sulphate-polyacrylamide gel electrophoresis band as judged by silver staining and represented about 25% of the total P450 applied to the column; total recoveries were usually more than 80%. Comparison with the molecular weights and spectral, catalytic and immunological properties of P450 forms isolated according to established procedures indicated that the form isolated here using Chelating Superose comprises mainly P450 2B1 (PB-B). A method is described for fully automated, programmable column regeneration and sample runs.     

A conjoint multi metal-ion iminodiacetic acid monolith microfluidic chip for structural-based protein pre-fractionation

PDMS-based multichannel microfluidic chip was designed and fabricated in a simple approach using readily available tools. UV-initiated in situ polymerization of poly(2-hydroxy ethyl methacrylate-co-di(ethylene glycol) diacrylate-co-N,Nʹ-diallyl l -tartardiamide) in an Eppendorf tube was achieved within 40 min. This polymerization process was successfully translated to a microfluidic chip format without any further modifications. Iminodiacetic acid was successfully immobilized on aldehyde functional monoliths via Schiff base reaction and confirmed by FT-IR spectroscopy. Four transition metal ions (Co (II), Zn (II), Ni (II), and Cu (II)) were chelated individually on four IDA-monolith microfluidic chips. The conjoint metal-ion monolith microfluidic chip has displayed high permeability (9.40 × 10^–13 m²) and a porosity of 32.8%. This affinity microfluidic chip has pre-fractioned four human plasma proteins (fibrinogen, immunoglobulin, transferrin, and human serum albumin) based on their surface-exposed histidine surface topography. A protein recovery of approximately 95% (Bradford assay data) was achieved. The multimonolith microchip can be reusable even after three protein adsorption-desorption cycles.