Anti-Zhangfei antibody (ab28700)

Why does this antibody detect a doublet around 50 kDa? The other Zhangfei antibodies in your catalog detect bands of different molecular weights, and the predicted molecular weight of this protein is 38 kDa. Are there multiple isoforms of Zhangfei? What is this antibody detecting?

ANSWER:

Further to my previous message, our lab has retested this antibody and produced the attached results. Ab28700 primarily detects the Smile-L isoform. I hope this helps, please let me know if you need any additional information. All lanes : Anti-Zhangfei antibody (ab28700) at 1 µg/ml Lane 1 : Heart (Human) Tissue Lysate - adult normal tissue (ab29431) Lane 2 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466) Lane 3 : Kidney (Human) Tissue Lysate - adult normal tissue (ab30203) Lane 4 : Liver (Human) Tissue Lysate - adult normal tissue (ab29889) Lane 5 : 293T Embryonic Kidney cell line (Human) Whole Cell Lysate (ab106304) Lane 6 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate (ab52257) Lane 7 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate (ab28419) Lane 8 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) Lane 9 : PC3 (Human prostate cancer cell line) Whole Cell Lysate (ab3954) Lane 10 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab52956) Lysates/proteins at 20 µg per lane. Secondary Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution Performed under reducing conditions. Predicted band size : 30 kDa Exposure time : 2 minutes

Why does this antibody detect a doublet around 50 kDa? The other Zhangfei antibodies in your catalog detect bands of different molecular weights, and the predicted molecular weight of this protein is 38 kDa. Are there multiple isoforms of Zhangfei? What is this antibody detecting?

ANSWER:

Thank you for contacting us. I apologize for the delay in my reply. Our lab provided the following explanation for the WB data:

This seems to be a challenging target; in Biochem. J. (2008) 416 (463–473) they state:

“SMILE (SHP-interacting leucine zipper protein) was previously designated as ZF (Zhangfei). SMILE gene generates two isoforms [SMILE-L (long isoform of SMILE) and SMILE-S (short isoform of SMILE). ZF is only a partial clone of CREBZF, which encodes protein with a predicted molecular mass of 37.2 kDa. Since ZF is a member of the bZIP family, we named CREBZF as SMILE.

(A) schematic of SMILE-L (CREBZF) and SMILE-S (Zhangfei/ZF).

(B) Exogenous expression of human wild-type SMILE and ZF in HEK-293T cells using the pcDNA3 vector. WB with SMILE-specific antibody.

(E) Expression of SMILE protein in cell lines (left-hand panel) and mouse tissues (right-hand panel).

To summarize, there is a long isoform, 50–60 kDa (SMILE-L), and a short isoform, 40 kDa (SMILE-S = ZF), and our ab seems to recognize the SMILE-L form.

I hope this helps to clarify the results. If you are not satisfied with this antibody, I would be happy to offer you a free of charge replacement, credit, or refund. Please let me know your original order number and how you would like to proceed and I would be happy to assist you further.