Note
L00436 interferes with IgG in mouse/rat serum and is not suitable for detecting mouse/rat serum samples.
| Product Description |
His tag is successive histidine
(H) residues and there are mainly three forms: HHHHHH (6 x His), HHHHH (5 x His),
HHHH (4 x His). Due to its small size, less interfere in protein folding, weak immunogenicity,
His tag is the most dominant tag, which is widely used in recombinant protein expression.
The DNA sequence which codes for His tag, is usually constructed at N-terminus
or C-terminus of variety of expression plasmids. Since His tag has high
affinity for Ni2+ ions, His-tagged proteins can be easily purified from
bacteria, yeast and mammalian cell expression system by Ni2+-resin chromatography.
Anti-His tag antibody is a useful tool for the identification of His-tagged
proteins with various methods such as western blot, immunoprecipitation and flow
cytometry.
GenScript His
Tag ELISA Detection Kit, a 1.5 hour competition
ELISA, is developed for rapid and high
throughput detection of His-tagged proteins. There are several potential usages:
●Quickly identify the
presence of His-tagged proteins in samples.
●Optimize protein
expression by monitoring the His-tagged proteins level.
●High throughput screening of
stable cell lines expressing His-tagged proteins.
This kit is
based on competitive ELISA method. His
Tag Plate in the kit is a 96-well microtiter plate coated with a His-tagged
protein with the molecular weight of 12.7 kDa. The plate, which comprises of 8
wells x 12 strips, is demountable. When Anti-His Monoclonal Antibody and His-tagged
proteins are added to the well , the coated His-tagged proteins compete with
His Tag Standard (His-tagged protein with the molecular weight of 11.3
kDa) in solution or His-tagged protein in sample to
interact with Anti-His Monoclonal Antibody. The higher the concentration of the
His-tagged protein in solution is, the less the antibody bound to the plate
will be. The Anti-His Monoclonal Antibody used in the kit is a mouse anti His tag
monoclonal antibody (GenScript A00186).
Antibody Tracer, which is a horseradish peroxidase (HRP) conjugated Goat anti mouse IgG, is used for
enzyme reaction. His-tagged
protein, Anti-His Monoclonal Antibody and antibody tracer form a complex. Other unbound
molecules can be removed by washing solution. The antibody tracer reacts with
TMB substrate to develop blue product that turns yellow immediately when the
Stop Solution is added, which can be measured by microplate reader at 450 nM.
In optimized
test condition, each absorbance value is indicated to the individual His-tagged
proteins amount in solution. His-tagged
protein standards of known concentration and the corresponding absorbance values are used to construct a standard
curve. With the standard curve, His-tagged protein amount present in the
unknown sample can be calculated by transforming its absorbance value. |
| Sensitivity |
1 ng/ml His-tagged proteins |
| Detection Range |
1 ng/ml~729 ng/ml |
| Test Samples |
N-terminal/C-terminal/internal His-tagged proteins4 x His/5 x His/6 x His-tagged proteins
Mammalian, yeast and bacteria cell lysates or cell supernatant |
| Conveniency |
Provide all reagents required for test
Complete test within
1.5 hours |
| Kit Contents |
| Component |
Quantity |
Part. No |
| His Tag Plate |
1 plate (8 wells x 12 strips) |
436-80 |
| Anti-His Monoclonal Antibody |
6 ml |
436-20 |
| Antibody Tracer |
12 ml |
436-30 |
His Tag Standards
(0,1,3,9,27,81,243,729 ng/ml) |
1 ml per each standard |
436-11,12,13,14,15,16,17,18 |
| Assay Diluent |
60 ml |
436-60 |
| 20 × Wash Solution |
40 ml |
436-70 |
| TMB Substrate |
12 ml |
436-40 |
| Stop Solution |
6 ml |
436-50 |
| His Tag Standard Stock (10 μg/ml) |
500 μl |
436-10 |
| Plate Sealer |
2 |
N/A |
| User Manual |
1 |
N/A |
|
| Reagents compatibility |
Tolerable
with various reagents at certain concentration |
| Storage |
The unopened kit is stable for
at least 12 months if stored at 2-8 °C
and the opened kit may be stable for up to 1 month at 2-8 °C. Do not freeze the kit. |
Concentration gradient represents X, OD450 value represents Y, all X values will be transformed to Log(X) to create the standard curve which was calculated by 4PL (Figure 1). The operator can get log values of sample concentration by inputting the OD450 value of unknown samples. By formula X=10X, sample concentration can be calculated.
For Research
Use Only. Not for Use in Diagnostic Procedures.
