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Finkelstein Lab Wiki

Epitope Tags for Labeling Proteins

We typically use fluorescent antibodies to visualize our epitope-tagged proteins.

Contents

Section titled “Contents”
  1. Recommended constructs
  2. TwinStrep Tag
  3. FLAG tag
  4. HA tag
  5. AviTag (biotin) tag
  6. V5 tag
  7. Sequence & Cloning
  8. Protein Expression
Section titled “1. Recommended constructs”

The following epitope tag / antibody combinations have worked well. I recommend creating the following three constructs in parallel:

  • 3xFLAG
  • 3xHA
  • AviTag
  • TwinStrep Tag (IBA)
  • V5 Tag

If you believe that the 3x tags may interfere with function, try making the 1x variants.

2. TwinStrep Tag

Section titled “2. TwinStrep Tag”

This is codon-optimized for E. coli:

5'-TGGAGCCATCCGCAATTTGAGAAAGGTGGTGGGAGCGGTGGTGGGTCTGGCGGCTCAGCCTGGTCCCATCCCCAGTTTGAAAAA-3'
WSHPQFEKGGGSGGGSGGSAWSHPQFEK

Reference: Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants

More information: IBA Life Sciences Twin Strep Tag PDF

3. FLAG tag

Section titled “3. FLAG tag”

Epitope sequence (either N- or C-terminal):

3xFLAG
5'-gactacaaagaccatgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaag-3'
DYKDHDGDYKDHDIDYKDDDDK


1xFLAG:
5'-GACTACAAGGACGACGATGACAAA-3'
DYKDDDDK

reference: [Sigma 3xFLAG system](http://www.sigmaaldrich.com/life-science/molecular-biology/cloning-and-expression/vector-systems/mat-system-vectors/3x-flag.html#CMV Vectors)

Use the anti-FLAG M2 monoclonal antibody from Sigma (F3165 PDF). The M2 antibody will label both N- and C-terminal epitopes. The M1 isolate requires Ca2+ and doesn’t label both N- and C-terminal fusions. Make sure the storage buffer does not contain BSA or other additives that may interfere with downstream QDot conjugation.

4. HA tag

Section titled “4. HA tag”

Epitope sequence (either N- or C-terminal):

3xHA
5'-TACCCATACGATGTTCCAGATTACGCTGGCTATCCCTATGACGTCCCGGACTATGCAGGATCCTATCCATATGACGTTCCAGATTACGCT-3'
YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA


1xHA:
5'-TACCCATACGATGTTCCAGATTACGCT-3'
YPYDVPDYA

reference: http://www.origene.com/destination_vector/PS100067.aspx PDF

Use the polyclonal anti-HA antibody from Immunology Consultants Lab (RHGT-45A (dead link)). Make sure the storage buffer does not contain BSA or other additives that may interfere with downstream QDot conjugation.

5. AviTag (biotin) tag

Section titled “5. AviTag (biotin) tag”

The AviTag was first identified by http://avidity.com PDF. Some proteins come out with >90% in vivo biotynilation efficiency in BL21s. However, if the tag is occluded or there is insufficient biotin ligase in the cells, biotinylation efficiency may be significantly lower. In that case, you can try a strain that over-expresses biotin ligase (BirA) or attempt in vitro biotinylation. See http://avidity.com PDF for more information.

6. V5 tag

Section titled “6. V5 tag”

peptide: GKPIPNPLLGLDST

3XV5:
(GKPIPNPLLGLDSTAAGKPIPNPLLGLDSTAAGKPIPNPLLGLDST)
Codon optimized for H. sapiens, with juggling. H. sapiens and S. frugiperda [Sf9; Sf21 cells] share very similar codon usage:
5’- GGGAAGCCGA TCCCGAACCC GCTGCTGGGG CTGGATAGCA CGGCCGCCGG CAAACCCATT CCCAATCCCC TGCTGGGCCT GGACAGCACC GCCGCCGGCA AGCCCATCCC CAACCCCCTG CTGGGCCTGG ATAGCACC-3'


E. coli (high expression) optimized (from IDT):
5'-GGCAAACCGA TCCCGAACCC GCTGCTGGGT CTGGATTCCA CGGCTGCCGG TAAACCGATC CCAAACCCAC TGCTGGGTCT GGATTCTACC GCTGCGGGCA AACCGATTCC GAATCCGCTG CTGGGCCTGG ACTCTACC-3'

7. Sequence & Cloning

Section titled “7. Sequence & Cloning”

C-terminal sequence:

5'-ggtggcggtctgaacgacatcttcgaggctcagaaaatcgaatggcacgaataa-3'


GGGLNDIFEAQKIEWHE*

reference: https://www.avidity.com/t-technologyvector-c.aspx PDF

N-terminal sequence:

5'-atgtccggcctgaacgacatcttcgaggctcagaaaatcgaatggcacgaa


MSGLNDIFEAQKIEWHE

reference: https://www.avidity.com/t-technologyvector-n.aspx PDF

NOTE: Try to keep this tag at the very N- or C-terminus of your protein. I’ve noticed <10% in vivo biotinylation efficiency when this epitope tag is sandwiched between an N-terminal His6 tag and my protein.

8. Protein Expression

Section titled “8. Protein Expression”

We’ve obtained efficient in vivo biotinylation (approaching 90%) by following the Avidity protocol:

Alternatively, you can add biotin directly to your cells (BL21, HMS174, etc) when you’re inducing your protein. The biotinylation efficiency is easily tested via an SDS-PAGE gel-shift assay (see Ilya).