
S-tag is the name of an oligopeptide derived from pancreatic ribonuclease A (RNase A). If RNase A is digested with subtilisin, a single peptide bond is cleaved, but the resulting two products remain weakly bound to each other and the protein, called ribonuclease S, remains active although each of the two products alone shows no enzymatic activity. The N-terminus of the original RNase A, also called S-peptide, consists of 20 amino acid residues, of which only the first 15 are required for ribonuclease activity. This 15 amino acids long peptide is called S15 or Stag.The amino acid sequence of the S-tag is: KETAAAKFERQHMDS conjugated to KLH. S- Tag antibody can recognize C-terminal, internal, and N-terminal S-tagged proteins.
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答:可能的原因有:
1、目的蛋白有多個修飾位點,本身可以呈現多條帶,建議查閱文獻或進行生物信息學分析,獲得蛋白序列的修飾位點信息,通過去修飾確定蛋白實際大小;
2、樣本處理過程中目的蛋白發生降解,建議加入蛋白酶抑制劑;樣本處理時在冰上操作;
3、雜蛋白多,建議處理目的蛋白;
4、抗體特異性不強,建議使用特異性強的抗體;
5、抗體孵育時間過久,建議減少抗體孵育時間;
6、二抗與抗原有交叉反應,建議選擇合適的封閉物;
7、二聚體或多聚體存在,建議增加蛋白質變性過程及強度;
8、底物顯色與曝光時間過長,建議縮短顯色及曝光的時間。
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