Magnetocapture of abalone transcription factor NF-kappaB: a new strategy for isolation and detection of NF-kappaB both in vitro and in vivo.
Rel\NF-kappaB plays an important role in the immune response. Recently, a homologue of Rel\NF-kappaB transcription factor was identified from a gastropod abalone, Haliotis diversicolor supertexta, named Ab-Rel. We present here a new strategy, so called magnetocapture, based on magnetic separation that was developed to isolate and detect this abalone transcriptional factor. With this method, Ab-Rel was successfully isolated from abalone haemocytes and the recombinant Ab-Rel protein from an insect cell line. Western blot analysis was performed to verify the effectiveness of this magnetocapture assay. Since only the activated NF-kappaB can be captured and separated from the whole cell extracts, this method was simultaneously used to detect the NF-kappaB activity in vivo, as well as the biological activity of the recombinant protein in vitro. In addition, several proteins which might interact with the abalone NF-kappaB were together isolated, suggesting that the magnetocapture method would be useful for identifying interaction molecules. These results illustrated that the magnetocapture strategy is rapid, reliable and versatile, providing a promising tool for studying Rel\NF-kappaB protein.
Jiang
YuSheng
Y
College of Animal Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, PR China.
Wu
Xinzhong
X
Zhang
Yang
Y
D000818:Animals; D002474:Cell-Free System / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D049851:Gastropoda / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006434:Hemocytes / Q000737:chemistry* / Q000378:metabolism; D009036:Moths / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D016328:NF-kappa B; D011994:Recombinant Proteins