• Immunoaffinity purification on columns conjugated to the antigen
• Protein G purification
• Protein A purification
• Ion exchange or size exclusion chromatography

Advantages of Custom Peptides from Krishgen Biosystem:
• Stringent quality standard. Certificate of analysis including MS and HPLC analysis is included in each order;
• Reasonable pricing. Bulk pricing is available;
• Purities from crude to 98% to fit you needs;
• A variety of special amino acids and modifications available;
• Peptides can be pre-aliquoted for your convenience.

Length of Sequence
The purity of a crude peptide typically decreases as the length increases. Peptides with sequences less than 15 residues can typically be made without difficulty. In addition, peptides of 10-15 residues in length are generally ideal for raising antisera.

Amino Acid Composition
To achieve high solubility, it is highly recommended that peptide design maintain a hydrophobic amino acid content of less than 50% and ensure that there is one charged residue for every five amino acids. In addition, peptides containing multiple cysteine, methionine or tryptophan residues are also difficult to synthesize mostly because these residues are susceptible to oxidation and hydrodgen bonding. Finally, beta-sheet formation during synthesis may cause a high degree of sequence deletion in the final product. This problem can be avoided by selecting sequences that do not contain multiple or adjacent residues comprised of valine, isoleucine, tyrosine, phenylalanine, tryptophan, leucine, glutamine, and threonine. If sequences cannot be chosen to avoid stretches of these residues, it often helps to break the pattern by making conservative replacements.

• Glycopeptide mapping
• Release of N- and O- linked glycans from the glycoprotein or glycopeptides
• Fluoroscent labeling of released glycans prior to chromatography or Mass Spectrometry
• Oligosaccharide mapping of fluorescently labeled glycans
• Oligosaccharide mapping of non-labelled glycans by HPAE-PAD
• Mass spectrometric analysis (including MSn fragmentation analysis) of derivatized and native glycans and glycopeptides by ion trap MS.

• Determination of the presence of carbohydrate impurities e.g. glucose contamination of a sample from purification column matrices or dialysis systems.
• An initial investigation prior to more detailed analysis of the glycosylation.
• Screening of glycoproteins manufactured using different cell lines.

• Pilot study to optimize monosaccharide analysis of samples with formulation buffer.
• Release of monosaccharides from intact glycoproteins + buffer controls.
• Release of monosaccharides from glycans removed from glycoproteins.
• Analysis of released monosaccharides using HPAEC-PAD (high pH anion exchange chromatography with pulsed amperometric detection) or fluorescent HPLC.
• Relative quantitation of the monosaccharide composition.

• An initial investigation prior to more detailed analysis of the glycosylation of the product.
• Screening of glycoproteins. Sialic Acid Profiling involves the following steps –
• Release of Sialic acid residues from the glycoprotein by mild acid hydrolysis.
• Separation and quantitation of sialic acid residues using HPLC or High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
• Determination of non-human sialylation by relative quantitative determination of NeuAc:NeuGC ratio.

• Estimation and Determination of extinction coefficient – the method include molecular weight measurement by MALDI-TOF MS.
• • Total amino acids, including cysteine, tryptophan, hydroxyproline.
• Free amino acids, including asparagine and glutamine.
• Protein concentration may be calculated by relating extinction coefficient to Optical Density measurement, which is performed using a UV spectrometer at 280nm.

• Hydrolyze a protein to individual constituent amino acids.
• Label amino acids with a detectable UV-absorbing or fluorescent marker.
• Separate different types of amino acids by chromatography.
• Measure relative amounts of each amino acid type based on intensity of the detectable marker associated with the emergence of each type of amino acid from the chromatographic system.

Peptide mapping can also be used for:

• Disulfide bridge assignment
• N-terminal and C-terminal sequence confirmation (normally together with MS/MS sequencing)
• Screening and indentification of sites of post-translational modification (e.g. glycosylation, phosphorylation)
• Guiding the choice of signals for subsequent MS/MS or gas phase sequence analysis

The four major steps involved in the development of a peptide map are:
• Isolation and purification of the protein.
• Selective cleavage of the peptide bonds (chemical or enzymatic).
• Chromatographic separation of the peptides.
• Analysis and identification of the peptides - Mass spectrometry (MS) or Electrospray MS (ES-Ms) or MALDI-MS or online LC-MS.

Isoenzyme analysis involves the following steps-

• Suspension cells or detachment of attached cells
• Enzyme extraction
• Electrophoresis
• Statistics involving calculation of migration distances, and comparison of ratios with the expected ones.

Determination of the isoenzyme gel electrophoresis banding pattern is carried out commonly for the following enzymes –
• Glucose - 6 - Phosphate Dehydrogenase (G6PD)
• Lactate Dehydrogenase (LD)
• Malate Dehydrogenase (MD)
• Nucleoside Phosphorylase (NP)
• Aspartate Aminotransferase (AST)
• Peptidase B (PepB