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Immunofluorescence analysis of HisG was done on HEK-293 cells transiently overexpressing HisG-RPS6 and untransfected cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with HisG Mouse Monoclonal Antibody (R940-25) at 2 µg - 4 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Rabbit Anti-Mouse IgG Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing cytoplasmic localization of HisG. Panel e shows HEK-293 untransfected cells. The images were captured at 20X magnification.
|Tested species reactivity||Tag|
|Published species reactivity||Not Applicable|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||6x His synthetic peptide: -His-His-His-His-His-His-Gly|
|Contains||0.01% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay Dependent|
|Immunofluorescence (IF)||2-4 µg/ml|
|Immunoprecipitation (IP)||Assay Dependent|
|Western Blot (WB)||1:500-1:2000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
This antibody is designed to specifically detect recombinant proteins containing the seven amino acid 6xHis-Gly epitope. This epitope is present in many expression vectors encoding an N-terminal 6xHis tag. The HisG antibody recognizes the following sequence: -His-His-His-His-His-His-Gly.
This product contains enough material for 25 Western blots. It has been tested against purified Positope™ control protein (100ng). The Positope™ control protein is a 53 kDa recombinant protein that contains seven epitope tags, including His(C-term), HisG, c-myc, and V5. For Western blot, dilute in PBS containing 0.05% Tween-20 and 5% nonfat, dry milk (PBSTM).
Using chemiluminescence as the detection method, no cross-reactivity has been observed in bacterial lysates. In mammalian lysates, a few cross-reactive proteins have been observed upon overexposure of blots.
This antibody has also been used successfully to immunoprecipitate fusion proteins that contain the 6xHis tag.
Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. Expression of recombinant proteins in E. coli as a fusion protein with neighboring histidine residues is one of the most popular methods of epitope tags. The affinity of the histidine-tag motif to Ni2+ by chelation is strong and selective enough to enable purification of the protein to homogeneity by affinity chromatography on a Ni2+-NTA adsorbent.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
The balance between two isoforms of LEF-1 regulates colon carcinoma growth.
R940-25 was used in western blot to elucidate aberrant LEF- activation in colon cancer.
|Wang SH,Nan KJ,Wang YC,Wang WJ,Tian T||BMC gastroenterology (12:null)||2012|
Endothelial induction of fgl2 contributes to thrombosis during acute vascular xenograft rejection.
R940-25 was used in western blot to test if induction of fgl2 on the vascular endothelium of xenografts contributes to thrombosis associated with acute vascular rejection
|Ghanekar A,Mendicino M,Liu H,He W,Liu M,Zhong R,Phillips MJ,Levy GA,Grant DR||Journal of immunology (Baltimore, Md. : 1950) (172:5693)||2004|
Punctin, a novel ADAMTS-like molecule, ADAMTSL-1, in extracellular matrix.
R940-25 was used in western blot to describe the localization and composition of Punctin (ADAMTSL-1)
|Hirohata S,Wang LW,Miyagi M,Yan L,Seldin MF,Keene DR,Crabb JW,Apte SS||The Journal of biological chemistry (277:12182)||2002|
|Not Applicable||Not Cited||
A novel single amino acid deletion caspase-8 mutant in cancer cells that lost proapoptotic activity.
R940-25 was used in immunoprecipitation to characterize a novel caspase-8 mutant, Delta Leu62casp-8
|Liu B,Peng D,Lu Y,Jin W,Fan Z||The Journal of biological chemistry (277:30159)||2002|
Elevated retinoic acid receptor beta(4) protein in human breast tumor cells with nuclear and cytoplasmic localization.
R940-25 was used in immunocytochemistry to describe the expression and structure of human RARbeta(4)
|Sommer KM,Chen LI,Treuting PM,Smith LT,Swisshelm K||Proceedings of the National Academy of Sciences of the United States of America (96:8651)||1999|
6xHis-Gly; 6xHis-Glycine; His G; His Tag; Histidine Epitope Tag; Histidine Tag