GeneTel#M20001

His-Tag (2A8)
Mouse mAbOrders877-248-4316
Info@genetel-lab.com

Support100 μl (50 Western mini-blots)

877-248-4316
Info@genetel-lab.com

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BACKGROUND
Plasmid vectors for the expression of coding regions of eukaryotic
genes in bacterial, insect and mammalian hosts are in common
usage; such expression vectors are frequently used to encode hybrid
fusion proteins consisting of a eukaryotic target protein and aspecialized region designed to aid in the purification and visualizationof the target protein. A system that has proven to be very successful
relies on the insertion of a six histidine (His6) sequence in the N++
terminus of the encoded protein, allowing for efficient coupling to Ni chelating resins and purification by single step affinity
chromatography. This polyhistidine sequence can then be removedby specific cleavage at sites recognized by enzymes such as
thrombin or enterokinase, permitting the separation of the target
protein from the polyhistidine tag. Visualization of such fusion
proteins can be achieved by utilizing antibodies generated against
specific peptide sequences downstream from the multiple cloningsite.
REFERENCES
1. Maniattis, T., et al. 1982. Molecular Cloning. Cold Spring
Laboratory, Cold Spring Harbor, NY.
2. Hochuli, E. 1988. Large-scale chromatography of recombinant
proteins. J. Chromatog. 444: 293-302.
SOURCE
This monoclonal antibody is produced by immunizing mice with a 6×
His synthetic peptide (KLH-coupled).
SPECIFICITY
His-Tag (2A8) Mouse mAb detects over-expressed or recombinant
proteins containing the 6× His epitope tag.
STORAGEStore at -20°C. Stable for one year from the date of shipment.
REACTIVITYAllISOTYPE
Mouse IgG1IMPORTANTUse an anti-MOUSE secondary antibody to detect the 2A8 antibody.
RECOMMENDED ANTIBODY DILUTIONS
Western blotting
1:5000Immunofluorescence
1:2000Immunoprecipitation
1:100
ELISA
1:2000* For Western blots, incubate membrane with diluted
antibody in 5% w/v nonfat dry milk, 1× TBS, 0.05% Tween20 at 4°C with gentle shaking, overnight.

rev. 12/09

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APPLICATION DATA
kDa 1

2

170130-kDa 1

3- His-Tagged

957255-Protein

1309572-2

3

4- His-Tagged
Protein55-43-

43-

Western blot analysis of overexpressed His-tagged protein in
293T cell lysate, using His-tag(2A8) Mouse mAb. The antibodydilutions are1:2000 (lane 1), 1:5000(lane 2) and 1:10000 (lane 3). Each

2A8 can specifically detect 1ng ofrecombinant protein. There is 6ng, 3 ng, 1 ng, and 0 ng Histagged recombinant protein inlanes 1-4, respectively, addedwith 20 μg of 293T cell lysate.

IF analysis of 293T cells transfected (left) with a His-tagged protein anda mock transfection (right, using the same protein without the His tag),using His-Tag (2A8) Mouse mAb at a 1:2000 dilution.
kDa 1
17013095725543-

2- His-TaggedProtein- IgG heavy chain3426-- IgG light chain

IP of extracts from 293T cells
transfected (lane 1) with a Histagged protein and a mocktransfection (lane 2, using the same
protein without the His tag), usingHis-Tag (2A8) Mouse mAb andprobed on Western blot using theCOMPANION PRODUCTS#M20002 Myc-Tag (19C2) Mouse mAb#M20003 HA-Tag (26D11) Mouse mAb#M20004 GFP-Tag (7G9) Mouse mAb#M20007 GST-Tag (12G8) Mouse mAb#M20008 DYDDDDDK-Tag (3B9) Mouse mAb (Binds tosame epitope as Sigma’s Anti-FLAG® M2 Antibody)

For in vitro research use only and not intended for use in