Desalting protocols

Protocol 2.11 Automated desalting protocol for ESI-MS analysis of intact proteins... 

Purify the reduced IgG from excess 2-mercaptoethylamine and reaction by-products by dialysis or gel filtration using a desalting resin. All buffers should contain 1-10 mM EDTA to preserve the free sulfhydryls from metal-catalyzed oxidation. The sulfhydryl-containing half antibody now may be used in conjugation protocols that use —SH-reactive heterobifunctional crosslinkers (Chapter 5, Section 1). 

Remove excess reactant by gel filtration using a desalting resin with PBS, pH 7.4. Some protocols use a quenching agent to remove excess periodate prior to gel filtration. This can be done by adding glycerol to a final concentration of 0.1 M. 

Whatever the technique employed to purify the RNA, it is necessary to desalt and concentrate it prior to use in crystallization trials. Avery efficient way of achieving this is to use reverse-phase Sep-Pak columns that can be used on the bench (Waters Sep-Pak Cl8 Classic short-body). These are operated by gravity or using a syringe (Protocol 14.3). 

Dissolve the enzyme to a concentration of 1 - 2 mg/ml in Soln. A (if it is delivered in another buffer, dialyze against Soln. A). Mix 1 mg -galactosidase in Soln. A with 0.25 mg SMCC activated antibody (concentration about 1 mg/ml for activation see Protocol 4.1.3). Shake at RT for 2 h, dialyze or desalt on a Sephadex G-25 column against Soln. B and concentrate to about 2 mg/ml. Mix 9 vol. of the concentrated conjugate with 1 vol. Soln. C and store without further purification at 4 °C. 

Following recommended protocols, buffer exchange an anti-His antibody into 0.1 M sodium carbonate buffer (pH 9.3) using a PD-10 desalting column (Amersham Biosciences) to give an antibody concentration of 1 mg/mL (see Note 11). 

Desalt the fractions using a sepharose weak anion exchange column according to Protocol 23. 

Because of the favorable sorptive properties of the reversed-phase supports, batch adsorption and desorption can be a very effective way to desalt a chromatographed sample or to partially fractionate a peptide mixture during a purification procedure. For example, 1-2 gm of an oc-tadecyl silica packed into a silanized glass or plastic pipette can be used for the batch fractionation of small amounts of a crude peptide extract from tissues, such as the pancreas or pituitary, or from a synthetic experiment. A number of commercial products, such as the Waters Sep-Pak, have found use in this manner 10) as a purification or sample preparation aid. Protocols for batch extraction procedures on alkyl silicas have been discussed 17a,b) and applied to neuropeptides 10, 158, 166) and other hormonal peptides 88, 162, 167, 168). With these methods recoveries of peptides present in a tissue extract are generally higher than those found with classical fractionation techniques due in part to the fact that proteolytic degradation is minimized. 

The following protocol is intended for use with PD-10 desalting columns, manufactured hy Amersham Biosciences. As long as you work quickly, Desalting can he completed at room temperature. Otherwise, if you have access to a cold room, we suggest you complete the procedure at 4°C. 

Desalt sulfotyrosine peptide samples by binding to RP-C18 ZipTips according to the manufacturer s protocol. After washing with HPLC-grade water, elute peptides with 70% acetonitrile. 

Desalt the peptides by Cl 8 column, and then analyze them by LC/MS. An MS protocol is necessary for this work. 

This protocol describes a procedure for the identification of PTM by HNE to reveal protein targets and specific sites of covalent attachment. Identification is performed by combining proteolytic digestion followed by SPH enrichment and nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS).The modified proteins are subjected to reduction, alkylation, and subsequent digestion by a proteolytic enzyme. The peptides thus obtained are desalted and the substoichiometric quantities of HNE-modified peptides are fractionated from unmodified species using hydrazide-coated glass beads-based enrichment technique that selectively captures peptide carbonyls (as hydrazones) that are subsequently... 

Quantify number of units (see Protocol 2) of all fractions including the sample load and wash. Combine only the fractions eluted with Solution B that contain large amounts of oligonucleotide. Do not combine the load and wash fractions since they are not desalted. 

Typically, sample desalting by the methods above (see text and Protocol 1) is sufficient for fimher experiments to be performed. 

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