Western Blot Analysis of Transfected Mammalian Lysates From CHO cell lysates, 1 mg of protein was electrophoresed on a 4-20% tris-glycine-SDS gel, then transferred to a nitrocellulose membrane, and reacted with antiluciferase (A) or anti-c-myc (B) antibodies. Antibody binding was detected using chemiluminescence methods. The difference in molecular weights of the different detected proteins is due to the use of epitope and purification tags. Estimated molecular weights: pDual GC + Luciferase is 68 kDa, pCMV-Tag5### + Luciferase is 63 kDa, and luciferase protein is 61 kDa. The difference in chemiluminescent signal between pDual GC + Luciferase and pCMV-Tag5 + Luciferase (B) is probably due to the presence of three copies of the c-myc epitope in the pDUAL GC vector and only one copy of the c-myc epitope in the pCMV-Tag5 vector. The lower molecular weight band that is detected at about 40 kDa with the anti-c-myc antibody (B) was also detected in control cells that had not been transfected with an expression vector (data not shown) and is believed to be a cellular protein that reacts with the c-myc-antibody.
>> Footnotes
1.Shine, J. and Dalgarno, L. (1974), Proc. Natl. Acad. Sci. USA71: 1342-1346.
2.Kozak, M. (1986), Cell.44: 283-292.
3.Padgett, K.A. and Sorge, J.A. (1998), Strategies.10: 97-99.
CMV promoter: Use of the CMV promoter is covered under the U.S. Patent Nos. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation and licensed FOR RESEARCH USE ONLY.
T7 Promoter: U.S. Patent No. 4,952,496. For academic and non-profit laboratories, an assurance letter accompanies the sale of the products. For commercial laboratories, a research use license agreement must be entered into prior to purchase of the products.
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