Peptide desalting

Centrifuge at 2500 x g for 10 min at room temperature and discard the pellet. The peptides must be desalted to remove salts and urea from the digestion buffer. In our laboratory, peptide desalting is achieved by reverse-phase C18 Sep-Pak SPE cartridges (Waters). 

The time required for preparation of cultured cell lysate is about 60 min and measurement of protein concentration using BCA assay is 40 min. Protein reduction and alkylation take about 75 min protein digestion is conducted overnight (12-18 h). Peptide desalting and lyophUization need 30 min and 2-4 h (depending on the volume after elution), respectively. 

Melanotropin. [9034-42-8] (18-22 amino acids peptide), amorphous. Extract separated by ion-exchange on carboxymethyl cellulose, desalted, evapd and lyophilised, then chromatographed on Sephadex G-25. [Lande et al. Biochem Prep 13 45 1971.]... 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

Relative molecular mass distributions for components of biochemical and polymer systems can be determined with a 10% accuracy using standards. With biochemical materials, where both simple and macro-molecules may be present in an electrolyte solution, desalting is commonly employed to isolate the macromolecules. Inorganic salts and small molecules are eluted well after such materials as peptides, proteins, enzymes and viruses. Desalting is most efficient if gels with relatively small pores are used, the process being more rapid than dialysis. Dilute solutions of macro-molecules can be concentrated and isolated by adding dry gel beads to absorb the solvent and low RMM solutes. 

In 1952 Carsten (Cl) developed a method, which allowed him to isolate and characterize several lower peptides contained in normal and pathological urine. According to this procedure, urine was desalted on the Amberlite IR-100 column and the adsorbed substances washed out with 2 M ammonia solution. The eluate was then passed through the column of Amberlite IRA-400. This column retained the ampholytes and rejected the weak bases. The former were recovered by elution with 1 M hydrochloric acid and the eluate was subsequently fractionated on Dowex 50 resin with 2M and later 4M hydrochloric acid as the eluents. By applying two-dimensional paper chromatography to further analysis of... 

A two-dimensional technique involving initial separation by high voltage electrophoresis at pH 2.0 followed by chromatography is a useful means of separating similar amino acids and short peptides and does not require desalting or excessive purification of the sample (Figure 10.17). 

If UV transparency is an issue for sensitive detection, nonvolatile solvents have been used. These include phosphoric acid,152,53 and TEAP, 24 among others. The latter is preferred as it allows for pH adjustment (2.0-8) and is biocompatible in most in vitro biological systems. It has been proposed that the role of the added triethylamine (to phosphoric acid, formic acid, TFA, and acetic acid) is to cap the unreacted silanols present on the silica gel. This issue has been recently addressed in a short review. 54 The TEAP buffer has been used extensively for the isolation of natural products122 and synthetic peptides. 26 Preparatively, the TEAP buffers were found to be good first solvents to use as they are highly resolutive with a selectivity that is often different from that conferred by 0.1% TFA. Since excess TFA will be eliminated by lyophilization, it is used as the last purification and desalting step. 

Add desalted crosslinker to reduced peptide, and leave overnight. 

Add desalted crosslinker/carrier to reduced peptide and leave overnight at room temperature. 

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