Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5.
Rab GTPases, which belong to the Ras superfamily, represent a group of small molecular weight GTP binding proteins that are involved in various steps along the exocytic and endocytic pathways. We first identified mRabL5 (GenBank Accession No. NP_080349), a novel Mus musculus Rab-like protein, present as a Golgi-associated protein. Here we presented the results of the cloning, prokaryotic expression, purification, and polyclonal antibody production of the novel Rab-like protein. In order to obtain a specific antibody against mRabL5, we prepared two GST fusion proteins, full-length mRabL5 GST fusion protein and mRabL5 C terminus GST fusion protein, to immunize rabbits. Western blot analysis showed that both antibodies prepared against full length of mRabL5 and its C terminus, respectively, can recognize mRabL5 protein. Immunofluorescence of mRabL5 in NIH3T3 cells using the two antibodies showed its perinuclear clustering distribution pattern. The polyclonal antibodies preparation against mRabL5 provided a good tool for us to study the functional involvement of mRabL5.
Yang
Jie
J
Jiangsu Key Laboratory for Molecular & Medical Biotechnology, Life Science College, Nanjing Normal University, Nanjing 210097, China.
Guo
Shi-Ying
SY
Pan
Fei-Yan
FY
Geng
Hui-Xia
HX
Gong
Yi
Y
Lou
Dan
D
Shu
Yong-Qian
YQ
Li
Chao-Jun
CJ
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005982:Glutathione Transferase / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016366:Open Reading Frames; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D020691:rab GTP-Binding Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D020691:rab GTP-Binding Proteins