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Tested applications | WB IF IP |
Recommended Dilution | WB 1:2000 - 1:5000 IF 1:50 - 1:500 IP 1:50 - 1:100 |
Observed MW | Refer to Figures |
Immunogen | A synthetic peptide of His tag |
Storage Buffer | Store at -20℃. Avoid freeze / thaw cycles. Buffer: PBS with 0.02% sodium azide, 50% glycerol, pH7.3. |
Western blot analysis of over-expressed His-tagged protein in 293T cell using His antibody at different dilution. Each lane was loaded with 2 ug cell lysate.
Immunoprecipitation of over-expressed His-tagged protein in HeLa cell incubated using His antibody. A mock served as negative control and over-expressed HeLa cell lysate served as positive control.
Plasmid vectors for the expression of coding regions of eukaryotic genes in bacterial, insect and mammalian hosts are in common usage; such expression vectors are frequently used to encode hybrid fusion proteins consisting of a eukaryotic target protein and a specialized region designed to aid in the purification and visualization of the target protein. A system that has proven to be very successful relies on the insertion of a six histidine (His6) sequence in the N-terminus of the encoded protein, allowing for efficient coupling to Ni++- chelating resins and purification by single step affinity chromatography. This polyhistidine sequence can then be removed by specific cleavage at sites recognized by enzymes such as thrombin or enterokinase, permitting the separation of the target protein from the polyhistidine tag. Visualization of such fusion proteins can be achieved by utilizing antibodies generated against specific peptide sequences downstream from the multiple cloning site.
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