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Affinity resins

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). [Pg.536]

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). [Pg.19]

Purification using anti-FLAG M2 affinity resin 9... [Pg.39]

Nickel affinity chromatography was chosen as the primary purification technique because it is a fast and reliable one-step assay and purified complexes can often be used in downstream applications without the necessity of removing the polyhistidine tag. In addition, the polyhistidine tag is smaller than many other affinity tags targeted by commercially available affinity resins and, in most cases, does not seem to interfere with the structure and function of the recombinant protein. [Pg.58]

Approximately 1 mg of WCE is mixed with 100 /A of M2 anti-FLAG-affinity resin (Sigma) and incubated at 4° for 2 hrs with gentle rocking. An aliquot of 3% of the total protein used in each reaction is stored at —20° to provide the input reference sample in the subsequent analysis. [Pg.66]

Affinity resins m7GDP-agarose matrix for purification of His6-eIF4E was prepared as previously reported (Edery et al., 1988). For purification of... [Pg.307]

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample. Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.
The biotinylated glycans on the cell surfaces subsequently may be probed with (strept)avidin reagents to detect the azido-sialic acid modifications. Alternatively, the cells may be lysed and the glycoproteins isolated using an immobilized (strept)avidin or monomeric avidin affinity resin. [Pg.693]

Affinity driven molecular transfer (ADMT) system, 78 264-265 Affinity ligands, 6 390 types of, 6 393-394, 396t Affinity method, 10 339 Affinity resins, 20 197 Affinity-selected libraries, 72 515-517 Affymatrix GeneChip HFV PRT, 76 390 Aflatoxins, 72 84... [Pg.21]

Fig. 5 2D PAGE of CSF before and after depletion with Cibacron blue/protein G affinity resins... Fig. 5 2D PAGE of CSF before and after depletion with Cibacron blue/protein G affinity resins...
Boc-aminooxyacetic succinimide ester in DMF (10 equiv, 0.5 M) was added to commercial amino Spherilose affinity resin (1 equiv) preswollen in DMF for 3 h. The resin was washed with DMF and the Boc group removed by treatment with neat TFA (2 min). The resin was washed again with DMF and neutralized with 10% DIPEA/DMF, and washed again with DMF. After thorough washing with CH2a2, the derivatized solid support 15 was dried. [Pg.79]

Identification of the Specific Binding Proteins of Bioactive Small Compound Using Affinity Resins... [Pg.181]

Key words Affinity resins, Chemical shotgun, FK506, Valdecoxib, BenzensuHonamide, Methotrexate... [Pg.181]

Affinity resins bearing bioactive compound have been widely used for identification of the specific-binding proteins (1, 2). However it is still troublesome to identify those proteins using traditional technology. Requirement of high level of synthetic chemistry expertise sometime restricts its application, especially for nonsynthetic chemists. On the other hand, the competition method is not often effective due to the poor solubility of orally active compounds. Some methods to solve these problems will be shown here, exemplified by identifications of the known specific binding proteins without such restrictions. [Pg.181]

Overview of Synthesis of Affinity Resins Bearing Bioactive Compound... [Pg.184]

Preparation of Conventional and Novel Effective Affinity Resins of Natural Product... [Pg.184]

Fig. 2. Preparation of CSG affinity resins of compound without effective inherent functional groups after bioconversion using S9 mixture, (a) Preparation of CSG affinity resins of valdecoxib (5) that has no effective inherent functional groups for immobilization, (b) Capture of C0X2 from sheep placenta lysate (Fig. 2b upper). Due to previously reported structure-activity relationships (8), we believe that conversion of the methyl group to hydroxymethyl was achieved by the S9 treatment. Moreover carbonic anhy-drase type 2 (CA2) was also captured by these resins from THP1, a human leukemic cell line, lysates (Fig. 2b lower). CA2 has been recently reported to be specifically inhibited by valdecoxib at 43 nM (10). Fig. 2. Preparation of CSG affinity resins of compound without effective inherent functional groups after bioconversion using S9 mixture, (a) Preparation of CSG affinity resins of valdecoxib (5) that has no effective inherent functional groups for immobilization, (b) Capture of C0X2 from sheep placenta lysate (Fig. 2b upper). Due to previously reported structure-activity relationships (8), we believe that conversion of the methyl group to hydroxymethyl was achieved by the S9 treatment. Moreover carbonic anhy-drase type 2 (CA2) was also captured by these resins from THP1, a human leukemic cell line, lysates (Fig. 2b lower). CA2 has been recently reported to be specifically inhibited by valdecoxib at 43 nM (10).
Preparation of Affinity Resins of Valdecoxib by Chemical Shotgun Method... [Pg.187]

After removal of buffer, the resulting affinity resins were calmly mixed with 1 mL of rat brain lysate at 4°C for 60 min (tee Note 12). [Pg.187]

Capture of C0X2 or Carbonic Anhydrase II (CA2) by Chemical Shotgun Valdecoxib-Affinity Resins... [Pg.188]

The resulting supernatant was carefully removed, and mixed with another 10 pL of ftesh valdecoxib-affinity resin at 4°C, again for about 45 min. [Pg.188]

Two tubes including the same FK506-affinity resins (10 pL each) and two tubes including 1 mL of the same lysate from rat brain were prepared in advance. [Pg.189]

Lysate from rat brain (1.0 mL) was stirred gently with FK506-affinity resin (10 pL) at 4°C for about 40 min. [Pg.189]


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Factorial Design for the Preparation of Affinity Resins

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