![]() At 100kDa a protein of the same intensity will only contain 1 picomole. A moderately intense spot of 10kDa containing 100ng protein will have a concentration of 10 picomole. Note: Coomassie Blue G-250 detects protein by mass (from 10-30 ng per band). Other stains may be compatible but please contact us prior to use. The protein can be stained with Coomassie Blue R-250, Amido black, Ponceau S or Sypro Ruby. In general, best results are obtained using 2D gels blotted to PVDF. It should only be used for well stained protein gel bands of at least 10pmol and is not recommended for proteins that are greater than 60kDa. ![]() Note: Passive elution is generally less efficient than electroblotting and does not effectively extract most high molecular weight proteins. Gels should be stained with Coomassie Blue R-250 or G-250. Protein can be passively eluted from polyacrylamide in an overnight procedure for an additional fee. APAF recommends IEF strips pre-prepared by Bio-Rad or GE Healthcare as these do not present this problem. Ampholines used to create pH gradients can also contain amines and reduce the efficiency of sequencing. ![]() Moreover, samples containing urea should not be stored for long periods and should not be heated in order to avoid carbamylation. Urea solutions used in gel preparation or solubilisation should be deionised with ion-exchange resin if not made from ultra high purity urea. Cyanate ions form particularly at alkaline pH. Also, if the protein is to be extracted from SDS gels, urea used as an electrophoresis reagent must be very pure and should have no cyanate ions present as these will carbamylate the proteins and block them. Note: The N-terminus of proteins can react with formic acid to form a formyl N-terminus, thus artificially blocking the protein. An additional fee will be charged for clean-up. We ask that a minimum of 10 picomole be supplied if clean-up is required. If interfering buffers/salts are unavoidable it should still be possible to sequence the material after clean up. Buffers containing amines, such as Tris and glycine, and detergents should be avoided. Sample should be free from interfering buffers, salts and detergents. If there will be a solubility problem please contact us. The sample will be reconstituted in 0.1% TFA/20% acetonitrile unless specified otherwise. Nitrocellulose is not compatible with the Edman chemistry.Protein can be submitted dry, in solution, as an SDS or native polyacrylamide gel piece, or electroblotted to PVDF membrane.A minimum of 10 picomole should be supplied for gel samples or if clean-up is required. Samples should contain 2 picomoles or more of the protein/peptide to be sequenced.Pure proteins (>90%) usually generate easily interpreted data, but insufficiently purified protein mixtures may also provide useful data.įor further details or advice on this APAF service please contact us at N-Terminal Sequencing Sample Preparation Guideline Sample Preparation N-terminal sequencing utilises the well-established Edman degradative chemistry, sequentially removing amino acid residues from the N-terminus of the protein and identifying them by reversed phase HPLC. Long sequences of 50 amino acids or more are possible with this technique. N-terminal sequencing (also called edman sequencing) is most commonly used to identify unknown proteins, confirm protein identity and quality (often for quality control of recombinant proteins), and identify protein N-terminus and cleavage sites. N-terminal protein sequence information continues to play a significant role in modern structural and molecular biology. N-Terminal Sequencing uses a chemical process based on the technique developed by Pehr Edman in the 1950's Press the 'Space' or 'Enter' key to toggle the Faculty of Science and Engineering navigation Press the 'Space' key to toggle the Faculty of Science and Engineering navigation Faculty of Science and Engineering. Press the 'Space' or 'Enter' key to toggle the Faculty of Medicine, Health and Human Sciences navigation ![]() Press the 'Space' key to toggle the Faculty of Medicine, Health and Human Sciences navigation Faculty of Medicine, Health and Human Sciences.
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