PROGEN Tag Antibodies

PROGEN offers a number of specific primary antibodies recognizing the most frequently used protein tags in cell biological studies including protein-protein interactions, posttranslational modifications, expression dynamics or localization, to laboratory-scale or large-scale production of biotherapeutics.

  • available for most commonly used protein tags (His, DDDDK, GST, HA, myc, GFP & Strep)
  • high affinity to the respective protein tag
  • robust & highly specific detection of the fusion protein
  • suitable for e.g. western blot (WB), immunoprecipitation (IP), ELISA & immunofluorescence (IF)

 

 

Why Using a Protein Tag?

 

                                                                                                                                                                                                                                                                                                     The use of recombinant proteins for cell biological studies including protein-protein interactions, posttranslational modifications, expression dynamics or localization increased in recent years. To allow rapid identification, modification, production, isolation & purification of proteins/molecules suitable tools including antibodies specifically targeting the protein of interest are needed. A convenient molecular method is the fusion of known protein tags, with high affinity to a specific ligand, to a recombinantly expressed protein.

 

Potential benefits

 

  • insertion or improvement of immunoreactive properties 

  • improve solubility

  • increase expression levels

  • support protein folding

  • decrease proteolytic degradation

 

Applications

 

  • identification of specific proteins (e.g. WB)

  • visualization & localization studies (e.g. IF)

  • isolation & purification (e.g. affinity purification)

  • analysis of protein-protein interaction

Commonly Used Protein Tags & Antibodies

His (Polyhistidin) Protein Tag

  • length varies between 2 to 10 histidine residues but normally 6 (6-His/His6)
  • fused to the C- or N-terminus
  • usually no interference with structure and function of the fused protein
  • protein specific interference can be resolved by changing the location of the tag
  • purification by immobilized metal-affinity chromatography (IMAC)
  • expression systems are bacteria, yeast, mammalian and insect cells
  • wide range of commercially available expression vectors and reagents

anti-6-His-tag Antibody

  • mouse monoclonal IgG1 antibody (clone 6His)
  • specific recognition of the 6-His-tag
  • high affinity & robust detection
  • suitable for ICC/IF, IP & WB
  • also available in large size (100µg)

anti-DDDDK-tag (Flag) Protein Tag

The DDDDK sequence is part of the Flag-tag (patented by Sigma Aldrich).

  • consists of 8 aa (DYKDDDDK), barely affects protein structure or function
  • added to N- or C-terminus (slightly beneficial at N-terminus)
  • detection in vivo & in vitro, 3x FLAG systems improves detection
  • purification (non-denaturing e.g. by antibody-mediated affinity chromatography)
  • not suitable for purification of high amounts but for isolation with high purity
  • very stable & not degraded in cells

anti-DDDDK-tag Antibody

  • mouse monoclonal IgG 2b antibody (clone AP1501)
  • recognizes DDDDK of the so called Flag-tag
  • high affinity & robust detection
  • suitable for ICC/IF, IP & WB
  • also available in large size (100µg)

GST (Glutathione-S-transferase) Protein Tag

  • consists of 211 amino acids with a molecular weight of 26kDa
  • fused to the N- or C-terminus
  • non-denaturing, single-step purification using the high affinity to glutathione
  • investigation of DNA-protein- or protein-protein-interactions
  • available vector systems often include protease cleavage site between protein sequence and tag
  • increases solubility, stabilizes & protects fusion protein from proteolytic degradation
  • can upregulate gene expression through the support of translational initiation
  • suitable expression systems are E. coli, mammalian cells, yeast or insect cells

anti-GST-tag Antibody

  • mouse monoclonal IgG1 antibody (clone F50-3D12.2)
  • specific recognition of GST
  • high affinity & robust detection
  • suitable for WB
  • also available in large size (100µg)

 

 

 

HA (Hemagglutinin) Protein Tag

  • derived from the human influenza hemagglutinin molecule (aa 98-106)
  • consists of 8 aa with a molecular weight of around 1kDa
  • barely affects the protein structure or function
  • highly immunoreactive
  • fused to the N- or C-terminus of the target protein
  • isolation & purification under mild conditions
  • detection & characterization in vitro & in vivo
  • an internal location within the protein encoding sequence is possible

anti-HA-tag Antibody

  • mouse monoclonal IgG2b antibody (clone 12CA5)
  • specific recognition of the HA-tag
  • high affinity & robust detection
  • suitable for WB
  • also available in large size (100µg)

 

myc Protein Tag

  • small-size epitope tag of 11 aa (1,2 kDa) derived from c-myc gene
  • effective in bacteria, yeast, mammalian and insect cells
  • detection of target proteins & monitoring of expression levels in cells
  • multiple copies can increase the signal strength
  • increased specificity if enzyme-linked secondary antibody is used for detection
  • suitable for affinity purification
  • elution is performed at low pH which might influence protein activity

anti-c-myc-tag Antibody

  • murin monoclonal IgG1 antibody (clone 9E10)
  • specific recognition of the myc-tag 
  • high affinity & robust detection
  • suitable for WB, IP, ELISA & FACS
  • also available in large size (100µg)

Strep (Streptavidin) Protein Tag

  • octapeptide of approximately 1kDa
  • no interference with proper folding & bioactivity of the protein
  • fused to the C- or N-terminus
  • purification using either a biotinylated matrix or streptavidin columns
  • investigation of functional proteins including protein-protein interaction
  • seems to be stable against proteolytic cleavage
  • successfully expressed in bacteria, yeast, plants, mammalian & insect cells

anti-Strep-tag Antibody

  • mouse monoclonal IgG1 antibody
  • specific recognition of the strep II tag
  • high affinity & robust detection
  • suitable for WB
  • also available in large size (100µg)

 

 

 

                                                                                                                                                                                                                                                                                        

Crucial Considerations to Choose the Right Tag

 

 

                                                                                                                                                                                                                                                                                                          

                                   

 

                                                                                                                                                                                                                                                                                                     The intended application of the potential tag is an essential factor and restricts the number of suitable tags to be used. In this regard, it is important to decide either for a small-size or a large-size tag according to the application of interest. Smaller tags usually have less effects on structure, activity or characteristics of the tagged protein. While larger tags can be used to improve features of the target protein. A major concern of using larger tags however is the loss/alteration of the biological activity or increased toxicity of the tagged target protein. It might be necessary to remove large tags by either a specific protease recognition site between the tag and the target protein or sequence encoding for a protein with self-splicing capacities.

Another consideration is the location of the tag within the protein of interest. Most of the tags are preferrably placed at the C- or N-terminus of the target protein to avoid interference with active sites. The positioning of the tag at one of the termini moreover ensures the exposure of the tag to the surface of the protein in order to allow the interaction with its ligand (e.g. for purification). In general, the oligonucleotide encoding for a tag is prevalently inserted at the 5’ end of the gene of interest to ensure a good translational initiation. In some cases it might also be possible to choose a location inside the gene sequence.

In order to increase the sensitivity of a tag, especially small-size tags, it is possible to attach tandem copies increasing signal strength and reinforcing signal-to-noise ratio. It is also possible to use a tandem tag system combining features of different tag groups. This is mainly used in the Tandem-Affinity Purification (TAP) allowing the isolation of protein complexes and the reduction of non-specific background based on two consecutive purification steps.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               There are two standard methods for the attachment of a tag to your protein of interest. First, genomic tagging by the introduction of an oligonucleotide encoding for the respective tag into the genomic coding sequence of the target protein. Second, the more prevalent way, by the introduction of the target protein coding sequence into an expression vector containing the desired tag sequence. The majority of modern expression vectors for mammalian, yeast, insect or E.coli cells carry one or even multiple tags. The vector-based method comes with several advantages, e.g. it is less labor-intense and much easier compared to the genomic tagging. Furthermore, this method allows the discrimination between the wild-type protein (untagged) and the tagged protein in a cell for analysis.

 

 

                                                                                                                                                                                                                                                                                                                                                                                         

Attachment of a Protein Tag