What are the sample conditions for determining the protein N-terminus?
I. Requirements for N-terminal Protein Sample Types
1. Purity Requirements
Protein purity is recommended to be >90% (single band on SDS-PAGE) to avoid interference from N-terminal signals of other proteins.
2. Protein Quantity
Generally, ≥10 µg pure protein is required. If enrichment/chemical derivatization is involved, ≥50 µg is recommended.
3. Buffer Composition Restrictions
(1) Avoid high concentrations of Tris (interferes with N-terminal derivatization) or amine-based buffers;
(2) Avoid strong detergents such as SDS, NP-40, Triton X-100;
(3) High salt (>200 mM NaCl) and glycerol (>10%) should also be avoided;
(4) Optimal conditions are 20 mM HEPES or PBS, pH 7.4, low salt (<100 mM NaCl).
4. Other Processing Information
(1) If the sample has undergone reduction, alkylation, enzymatic digestion, or tag fusion (e.g., His-tag), it must be clearly stated, especially whether the N-terminal is naturally exposed;
(2) Information about the expression system (e.g., E. coli, mammalian, eukaryotic) must also be provided, as it affects N-terminal modifications (such as N-terminal formylation, acetylation, cleavage).
II. Requirements for C-terminal Protein Sample Preparation
1. Purity and Sample Amount
Protein purity is recommended to be >90%, with a quantity of ≥50 µg, as the C-terminal signal is relatively weak and requires a higher input amount to enhance detection success.
2. Buffer Requirements
(1) Avoid amine-based buffers (like Tris), strong detergents (SDS), high salt, or glycerol;
(2) It is recommended to use 20 mM HEPES, pH 7.4, under low salt conditions (<100 mM NaCl).
3. Protein State and Modification Information
(1) Clarify whether it is a naturally terminated C-terminal, whether it carries tags (such as His-tag, FLAG), and the expression system (E. coli expression often accompanies non-natural C-terminal);
(2) Common C-terminal modifications such as amidation or carboxymethylation can affect mass spectrometry recognition, and it is recommended to evaluate their possibility in advance.
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