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Purification & Detection

MAT-Tag Protein Expression

Combine IMAC Versatility with the Sensitivity of FLAG® Detection


The MAT-Tag™ System (metal affinity tag) utilizes a small, novel, seven amino acid (HNHRHKH) sequence created for IMAC purification of recombinant MAT-Tag™ fusion proteins and the high sensitivity detection of the antibody based FLAG® System.

  • Dual tagged fusion proteins
  • N- or C- terminal tagging configurations
  • Compatible to Nickel and Cobalt affinity resins, such as
  • Superior antibody detection

Data

Technical Posters

 

MAT-Tag™ Bacterial Expression Vectors
tac Promoter System
T7 Promoter System
T7 Promoter System with Enterokinase Recognition Site

MAT-Tag™ Mammalian Expression Vectors
Transient Expression

MAT-Tag™ Baculovirus Transfer Vectors

MAT-Tag™ Monoclonal Antibody



Data


FLAG-BAP-MAT-Tag fusion protein detected by western blot MAT-Tag- fusion protein affinity purified by IMAC visible by SDS-PAGE
Figure 1. Full-length FLAG-BAP-MAT-Tag™ fusion protein is detected on Western blots by monoclonal antbodies to the FLAG and MAT-Tag™ tags. Lysates from uninduced (lanes 2 and 5) and induced (lanes 3 and 6) E. coli cultures and ColorBurst Markers (lanes 1 and 4) were seperated by SDS-PAGE and blotted to nitrocellulose. The resulting blot was immunostained using either ANTI-FLAG® M2-HRP conjugate (lanes 1-3) or Anti-MAT-Tag™ monoclonal antibody (0.5 µg/ml) followed by Rabbit-Anti-Mouse IgG-HRP conjugate (lanes 4-6). The blots were developed and visualized with TMB Substrate. Figure 2. MAT-Tag™-tagged fusion protein expressed in E. coli can be affinity purified by IMAC. The coding region for GrpE was cloned into an E. coli expression plasimd, pFLAG-MAC, with an N-terminal FLAG tag and a C-terminal MAT-Tag™ tag. The fusion protein was expressed in E. coli strain BL21. A lysate (L, lane 2) was made in CelLytic B and separated by centrifugation into a pellet (P, lane 3) and a supernatant (S, lane 4) fraction. The supernatant was applied to a 1.0 mL HIS-Select HF Nickel Affinity Gel column and the flow through (F, lane 5), wash (1-3, lanes 6-8) and elution (1-4, lanes 9-12) fractions were collected. Samples of the fractions and ColorBurst Markers (lane 1) were seperated by SDS-PAGE and visualized by EZBlue staining.

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MAT-Tag™ Bacterial Expression Vectors

tac Promoter System

Vectors utilizing the strong tac promoter (a hybrid of the E. coli trp and lac promoters) offer protein expression levels in excess of 10 mg/L of culture when using IPTG as a de-repressor. These vectors can be used to express protein in any established E. coli host.

pTAC-MAT-Tag™-1

Cytoplasmic expression of N-terminal MAT-Tag™ fusion proteins under the tac promoter. Supplied with a pTAC-MAT-Tag-2+BAP Control Vector.

Product # Product Name Size
E5530 pTAC-MAT-Tag™-1 Expression Vector 10 µg
pTAC-MAT-Tag-1 Expression Vector
pTAC-MAT-Tag™-2

Cytoplasmic expression of C-terminal MAT-Tag™ fusion proteins under the tac promoter. Supplied with a pTAC- MAT-Tag™-2+BAP Control Vector.

Product # Product Name Size
E5405 pTAC- MAT-Tag™-2 Expression Vector 10 µg
pTAC- MAT-Tag™-2 Expression Vector

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MAT-Tag™ Bacterial Expression Vectors

T7 Promoter System

The pT7- MAT-Tag™ vectors offer the very strong T7/lac promoter. These expression vectors produce even higher yields of recombinant proteins than the tac promoter system. However, the T7 promoter is known for background (“leaky”) expression, which can be a drawback when recombinant proteins are toxic to the host cell. Therefore, Sigma’s vectors contain the lac operator (lacO) sequences immediately downstream from the promoter to reduce leaky expression. Unlike the tac promoter system, pT7 vectors must be expressed in hosts containing a source of the T7 polymerase such as (DE3) lysogenic strains.

pT7- MAT-Tag™-1 pT7- MAT-Tag™-1

Cytoplasmic expression of N-terminal MAT-Tag™ fusion proteins under the T7llac promoter. Supplied with a pT7- MAT-Tag™-2+BAP Control Vector.

Product # Product Name Size
E5780 pT7- MAT-Tag™-1 Expression Vector 10 µg
pT7- MAT-Tag™-2 pT7- MAT-Tag™-2

Cytoplasmic expression of C-terminal MAT-Tag™ fusion proteins under the T7llac promoter. Supplied with a pT7- MAT-Tag™-2+BAP Control Vector.

Product # Product Name Size
E5655 pT7- MAT-Tag™-2 Expression Vector 10 µg
pT7-FLAG- MAT-Tag™-1 pT7-FLAG- MAT-Tag™-1

Cytoplasmic expression of N-terminal Met-FLAG, C-terminal MAT-Tag™ dual tagged fusion proteins under the T7llac promoter. Supplied with a pT7-FLAG- MAT-Tag™-1+BAP Control Vector.

Product # Product Name Size
E5280 pT7-FLAG- MAT-Tag™-1 Expression Vector 10 µg
pT7- MAT-Tag-FLAG™-2 pT7- MAT-Tag-FLAG™-2

Cytoplasmic expression of N-terminal MAT-Tag™, C-terminal FLAG dual tagged fusion proteins under the T7llac promoter. Supplied with a pT7-FLAG- MAT-Tag™-1+BAP Control Vector.

Product # Product Name Size
E4905 pT7- MAT-Tag-FLAG-2 Expression Vector 10 µg

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MAT-Tag™ Bacterial Expression Vectors

T7 Promoter System with Enterokinase Recognition Site

The recognition sequence for enterokinase, Asp-Asp-Asp-Asp-Lys, is found at the C-terminal end of the FLAG® epitope tag. Removal of the FLAG is possible in all fusion proteins containing an N-terminal FLAG sequence. Dual tag fusion proteins may also be cleaved with enterokinase for removal of one or more tags, depending on the position of FLAG in the protein sequence.

pT7- MAT-Tag-FLAG™-1

Cytoplasmic expression of N-terminal MAT-Tag-FLAG dual tagged fusion proteins under the T7llac promoter. Supplied with a pT7-FLAG- MAT-Tag-1+BAP Control Vector.

Product # Product Name Size
E5155 pT7-MAT-Tag-FLAG-1 Expression Vector 10 µg
pT7-MAT-FLAG™-1
pT7-FLAG-MAT™-2

Cytoplasmic expression of C-terminal FLAG- MAT-Tag dual tagged fusion proteins under the T7llac promoter. Supplied with a pT7-FLAG- MAT-Tag™-1+BAP Control Vector.

Product # Product Name Size
E5030 pT7-FLAG- MAT-Tag™-2 Expression Vector 10 µg
pT7-FLAG-MAT™-2

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MAT-Tag™ Mammalian Expression Vectors
Transient Expression

CMV vectors contain the pMB1 (derivative of pBR322) origin for replication in bacterial cells, the β-lactamase gene for ampicillin resistance selection in bacteria, hGH, polyA, and the f1 origin.

pCMV-FLAG- MAT-Tag™-1

Transient cytoplasmic expression of N-terminal MeT-FLAG, C-terminal MAT-Tag™ dual tagged fusion proteins under the CMV promoter. Supplied with pCMV-FLAG- MAT-Tag™-1 MAPK1 Control Vector.

Product # Product Name Size
C5864 pCMV-FLAG- MAT-Tag™-1 Expression Vector 20 µg
pCMV-FLAG- MAT-Tag™-1
pCMV-MAT-FLAG™-1

Transient cytoplasmic expression of N-terminal-MAT-Tag -FLAG dual tagged fusion proteins under the CMV promoter. Supplied with pCMV-FLAG- MAT-Tag™-1 MAPK1 Control Vector.

Product # Product Name Size
C5989 pCMV-MAT-Tag-FLAG-1 Expression Vector 20 µg
pCMV-MAT-FLAG™-1
pCMV-FLAG- MAT-Tag™-2

Transient cytoplasmic expression of C-terminal FLAG- MAT-Tag™ dual tagged fusion proteins under the CMV promoter. Supplied with pCMV-FLAG- MAT-Tag™-1 MAPK1 Control Vector.

Product # Product Name Size
C6114 pCMV-FLAG- MAT-Tag™-2 Expression Vector 20 µg
pCMV-FLAG-MAT™-2

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MAT-Tag™ Baculovirus Transfer Vectors

pPolh-MAT-Tag™ are baculovirus transfer vectors used for producing Metal Affinity Tag (MAT-Tag™) fusion proteins in insect cells. The pPolh- MAT-Tag™ vectors contain the strong viral polyhedrin (polh) promoter for high-level expression of target genes during the very late phase of infection. The vectors also contain a high copy of bacterial origin of replication and an ampicillin resistance gene (ampr) for easy propagation in E. coli host cells.




pPolh- MAT-Tag™-1 Features

Feature Map Position
AcNPV Sequence (ORF 603) 1-1146
Recommended 5' primer sequence binding site 1079-1100
polh Promoter 1076-1145
MAT-Tag™ 1167-1187
MCS 1188-1246
Recommended 3' primer sequence binding site 1300-1320
M13 origin 2576-3229
poly A 1599-1604
AcNPV Sequence (ORF 1629) 1286-2629
β-lactamase (amp') 3616-4473
pUC ori 4624-5267

pPolh- MAT-Tag™-2 Features

Feature Map Position
AcNPV Sequence (ORF 603) 1-1146
Recommended 5' primer sequence binding site 1079-1100
polh Promoter 1076-1145
MCS 1148-1211
MAT-Tag™ 1212-1232
Recommended 3' primer sequence binding site 1295-1315
M13 origin 2571-3224
poly A 1594-1599
AcNPV Sequence (ORF 1629) 1281-2624
β-lactamase (amp') 3611-4468
pUC ori 4619-5262


Product # Product Name Size Application
T6699 pPolh-MAT™-1 Transfer Vector 20 µg Expression of N-terminal MAT-Tag™ fusion proteins in insect cells
T6574 pPolh-MAT™-2 Transfer Vector 20 µg Expression of C-terminal MAT-Tag™ fusion proteins in insect cells



MAT-Tag™ Antibody
Product # Product Name Size Characteristics Application
M6693 Monoclonal Anti-MAT™ antibody produced in mouse 200 µg Specificity: N-terminal and C-terminal MAT-Tag™ fusion proteins
  • Immunoprecipitation
  • Immunocytochemistry
  • Western blotting
  • EIA
Working Dilution 1 µg/ml by indirect Western blotting (chemiluminescent)


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