Anti·His Antibodies, BSA-free

For sensitive detection of His-tagged proteins with an RGS·His or HHHH epitope

Features

Highly specific detection of C-terminal, N-terminal and internal His tags
Highly sensitive and specific detection of RGS·His epitopes
Highly sensitive and specific detection of HHHHH epitopes

Useful Resources

QIAexpress Detection and Assay Handbook - (EN) QIAexpress® Data Sheet: RGS·His™ Antibody epitope - (EN)

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RGS·His Antibody, BSA-free (100 μg)

Cat. No. / ID: 34650

100 μg mouse anti·RGS(H)₄ (lyophilized, BSA-free, for 1000 ml working solution)

DKK 5,475.00

Tag

RGS·His

Tetra·His

Penta·His

Quantity

Anti·His Antibodies, BSA-free are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

Monoclonal mouse Anti·His Antibodies (6xHis antibodies and 4xHis antibodies), like the RGS·His Antibody, BSA-free, Tetra·His Antibody, BSA-free and Penta·His Antibody, BSA-free are used to detect recombinant protein carrying His tags. Each of the three variants detects a different His tag epitope. Like all QIAGEN mouse monoclonal antibodies, they are prepared under serum-free conditions, which guarantees the absence of viruses, mycoplasma and contaminating immunoglobulins.

Performance

Many different proteins have been detected, via their His tags, using QIAexpress Anti·His Antibodies (6xHis antibodies and 4xHis antibodies) (see table “Performance of Anti∙His Antibodies”). For the sensitivity of the three Anti·His Antibodies in detecting this panel of proteins see the figure “Sensitivity of QIAexpress Anti·His Antibodies”). Anti·His Antibodies enable identification of positive expression clones by colony blotting (see figure “Identification of positive expression clones by colony blotting”) and detection of His-tagged thioredoxin (see figure “His-tagged thioredoxin detected by Penta·His Antibody in yeast cells”). The antibodies are also ideally suited for use in immunohistochemistry procedures (see figure “Immunohistochemistry with Penta·His Antibodies”).

Performance of Anti∙His Antibodies

Feature	RGS·His Antibody, BSA-free	Penta·His Antibody, BSA-free
Detection	N-terminal His tags (containing the epitope RGSHHHH)	N-terminal, C-terminal and internal 6xHis tags
Sensitivity in dot blots*	0.5 ng	0.5 ng
Sensitivity in western blots*	2 ng	–
Isotype	Mouse IgG1	Mouse IgG1
Cross-reactivity	Negligible with crude E. coli, yeast, mammalian or insect cell lysates	Negligible with crude E. coli, yeast, mammalian or insect cell lysates
Form	Lyophilized	Lyophilized
Working solution	1000 ml	1000 ml

* Detection using chromogenic substrate. Sensitivity may vary with different proteins.

Proteins detected with QIAexpress Anti∙His Antibodies

	Protein	Tag location	Sequence
A	DHFR	N-terminus	MRGSHHHHHHGSVRPLNCIV..........
B	DHFR	C-terminus	.........VYEKKDGSRSHHHHHH
C	Thioredoxin	N-terminus	MRGSHHHHHHGSSDKIIHLT..........
D	TNF-α	N-terminus	MRHHHHHHQSTESMIRDV.........
E	TFIIAγ	N-terminus	MGSSHHHHHHSSGLVPRGSH.....
F	Chaperonin	C-terminus	..........AKIAKDGSRSHHHHHH
G	DNA polymerase	C-terminus	..........EDWLSAKERSHHHHHH
H	TFIIAαß	Internal	......QQQHQPQQQQHHHHHHHQQAQPQQTVP....

See figures

Sensitivity of anti·His antibodies

Identification of positive expression clones by colony blotting.

His-tagged thioredoxin detected by Penta·His Antibody in yeast cells.

Immunohistochemistry with Penta·His Antibodies.

Principle

The QIAexpress System provides materials for the expression, purification, detection and assay of 6xHis-tagged proteins. For detection, QIAexpress mouse monoclonal antibodies are produced under conditions that guarantee the highest purity and activity. Hybridoma cell culture is carried out under serum-free conditions, ensuring that the preparations are free of viruses, mycoplasma and contaminating immunoglobulins. Purification by adsorption chromatography is performed entirely at physiological pH, yielding preparations with the highest proportion of native antibody.

Procedure

The QIAexpress, Detection and Assay Handbook, supplied with all QIAexpress detection and assay products, contains detailed example protocols and guidelines for designing and performing assays.

Applications

QIAexpress Detection Systems, including RGS·His Antibody, BSA-free, Tetra·His Antibody, BSA-free and Penta·His Antibody, BSA-free allow efficient detection of His-tagged proteins and are ideal for many applications, including:

Colony, dot and western blotting procedures
Screening for positive expression clones
Monitoring expression levels and stability of His-tagged proteins
Immunoprecipitation and ELISA
Immunocytochemistry and immunohistochemistry

Comparison of Anti·His Antibodies

Features	QIAamp MinElute Virus Spin Kit	Tetra·His Antibody, BSA-free	Penta·His Antibody, BSA-free
Detection	Secondary antibody required	Secondary antibody required	Secondary antibody required
Epitope detected	RGSHHHH	HHHH	HHHH
Sensitivity in dot blots -chemiluminescent substrate-	10 pg	10 pg	10 pg
Sensitivity in dot blots -chromogenic substrate-	0.5 ng	0.5 ng	0.5 ng
Sensitivity in western blots - Chemiluminescent substrate	50 pg	50 pg	50 pg
Sensitivity in western blots -chromogenic substrate-	2 ng	2 ng	2 ng
Substrate for blot detection	Dependent on secondary antibody	Dependent on secondary antibody	Dependent on secondary antibody
Substrates for assay procedure	Dependent on secondary antibody	Dependent on secondary antibody	Dependent on secondary antibody
Tag	His tag	His tag	His tag

Anti-His Antibodies, BSA-free are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Supporting data and figures

Sensitivity of anti·His antibodies.

Detection of 6xHis-tagged proteins with Anti·His Antibodies (Tetra·His Antibody in the center). A: DHFR; B: DHFR; C: thioredoxin; D: TNF-α; E: TFIIAγ; F: chaperonin; G DNA polymerase; H: TFIIAαß; for tag location and sequences detected see table "Proteins detected with QIAexpress Anti·His Antibodies". Indicated amounts of pure 6xHis-tagged protein were applied to a nitrocellulose membrane, and detection was carried out with the Anti·His primary antibody indicated diluted 1/2000, followed by chromogenic detection with AP-conjugated rabbit anti-mouse IgG and NBT/BCIP.

Resources

Download Safety Data Sheets for QIAGEN product components.

For Anti·His Antibodies, Anti·His HRP Conjugates, Penta·His™ Alexa Fluor® Conjugates, Penta·His Biotin Conjugate, Ni-NTA Conjugates, Tag·100™ Antibody, Streptavidin–R-PE, 6xHis Protein Ladder, Ni-NTA HisSorb™ Strips and Plates

Publications

Localization of the gD-binding region of the human herpes simplex virus receptor, HveA.

Whitbeck JC; Connolly SA; Willis SH; Hou W; Krummenacher C; Ponce de Leon M; Lou H; Baribaud I; Eisenberg RJ; Cohen GH;

J Virol; 2001; 75 (1):171-80 2001 Jan PMID:11119586

Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme.

Dierks T; Schmidt B; Borissenko LV; Peng J; Preusser A; Mariappan M; von Figura K;

Cell; 2003; 113 (4):435-44 2003 May 16 PMID:12757705

Screening for soluble expression of recombinant proteins in a 96-well format.

Knaust RK; Nordlund P;

Anal Biochem; 2001; 297 (1):79-85 2001 Oct 1 PMID:11567530

A human cDNA expression library in yeast enriched for open reading frames.

Holz C; Lueking A; Bovekamp L; Gutjahr C; Bolotina N; Lehrach H; Cahill DJ;

Genome Res; 2001; 11 (10):1730-5 2001 Oct PMID:11591650

Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments.

Dirac-Svejstrup AB; Shorter J; Waters MG; Warren G;

J Cell Biol; 2000; 150 (3):475-88 2000 Aug 7 PMID:10931861

FAQ

Dissolve the lyophilized Anti-His Antibodies (100 µg) in 500 µl water per vial. The final concentration is 0.2 mg/ml. Dissolve the Anti-His Antibody Selector Kit antibodies (3 µg) in 15 µl water per tube (final concentration, 0.2 mg/ml).

-100

The table below lists the epitopes recognized by the Anti-His Conjugates and Anti-His Antibodies. 'H' designates a histidine residue, while 'X' designates any amino acid other than histidine. Note that individual 6xHis-tagged proteins are often recognized better by one Anti-His Antibody than by the others, possibly because of subtle differences in the exact conformation of the 6xHis tag and other parts of the protein in the vicinity of the tag. To choose the optimal antibody for your specific 6xHis-tagged protein and application, QIAGEN offers the Anti-His Antibody Selector Kit. The kit includes 3 µg of each of the three antibodies for direct comparison and economical selection of the one that gives the best results.

Epitopes recognized by QIAexpress Anti-His HRP Conjugates and Anti-His Antibodies

Peptide	Penta-His	Tetra-His	RGS-His
XHHHHHHX	+	+	–
XXHHHHHX	+	+	–
XHXHHHHX	–	+	–
XHHHHXHX	–	+	–
XRGSHHHHX	–	+	+
XRGSHHHHHHX	+	+	+
XHXHHXHX	–	–	–

Note that the presence of specific residues N- or C-terminal to the epitope is not required.

FAQ ID -170

Penta-His and Tetra-His Antibody bind to proteins that have 6xHis tags regardless of the surrounding amino-acids, even when the tag is partially hidden. Subtle differences in the antigen recognition sites of these antibodies mean that some 6xHis-tagged proteins are detected with higher sensitivity by one Anti-His Antibody than by the others (see figure below). Therefore, to achieve optimum results, we recommend comparison of these antibodies in parallel for new applications and proteins. QIAGEN offers the Anti-His Antibody Selector Kit for this purpose.

Sensitivity of QIAexpress Anti·His Antibodies

Note: RGS His Antibody regonizes the RGS(His)4 epitope that is found on proteins endcoded by most pQE vectors as well as several pRSETan pBlue BacHis -vectors from Invitrogen.

FAQ ID -172

Anti·His Antibody epitopes and dissociation constants

Antibody	Epitope	Dissociation constant K_d (M)*
RGS·His Antibody	RGSHHHH	1 x 10^–8 – 5 x 10^–8
Penta·His Antibody	HHHHH	5 x 10^–8 – 1 x 10^–9
Tetra·His Antibody	HHHH	1 x 10^–8 – 5 x 10^–8

* Dissociation constants were measured using surface plasmon resonance (BIACORE) technology. The exact value of K_d is dependent on the individual 6xHis-tagged protein.

FAQ ID -385

Yes, QIAGEN antibodies are glycosylated to a low extent. We did not determine whether this is N-glycosylation or O-glycosylation. Furthermore, we do not know, if the glycosylation pattern does change, since we do not measure it. Glycosylation does not have any impact onto the function of the antibody. If customers want to use the glycosylchains for coupling reactions we know that this is very difficult to do.

FAQ ID -334

This is done via epitope mapping. The antibody in question is first tested against different protein fragments and peptides. Once the amino acids detected by the antibody are determined, the surrounding amino acids can be exchanged to reveal the influence of the environment on the detection of a specific epitope.

FAQ ID -593

Please do not use buffer containing milk powder to block the Western Blot. This will reduce sensitivity. QIAGEN recommends use 3% BSA in TBS-buffer to block the membrane and for incubation of the Anti His Antibody on the membrane. For the binding of the secondary Antibody BSA does not sufficiently block and therfore 10% milk powder in TBS-buffer should be used at this step only. Alternatively, if alkali-soluble casein (Merck, Cat. No. 1.02241) is available in your country it can be used as a blocking reagent throughout the chemiluminescent detection protocol. Please see Table 9 of the QIAxpress Detection and Assay Handbook for additional information.

FAQ ID -1093

BSA stabilizes the RGS-His Antibody. We recommend to use the BSA-containing RGS-His Antibody for all standard procedures like dot blots, western blots, etc.. In cases where BSA may interfere with the application, e.g. coupling reactions, the RGS-His Antibody, BSA-free is recommended.

The Tetra-His and Penta-His Antibodies are stable without BSA, and therefore are only offered BSA-free.

FAQ ID -569

QIAGEN Anti·His Antibodies should be stored lyophilized until they are to be used. They can be stored lyophilized for 1 year at 2–8°C. In solution they can be stored for 3 months at 2–8°C or for up to 6 months in aliquots at –20°C. Avoid repeated freezing and thawing.

FAQ ID -194

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