DYKDDDDK Tag antibody

Catalog No. ABIN99294

Product Details anti-DYKDDDDK Tag Antibody

Target: DYKDDDDK Tag

Reactivity: Please inquire

Host: Rabbit

Clonality: Polyclonal

Conjugate: This DYKDDDDK Tag antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunoprecipitation (IP)

Sequence: DYKDDDDK

Characteristics: Concentration Definition: by UV absorbance at 280 nm

Immunogen: This antibody was purified from whole rabbit serum prepared by repeated immunizations with the Enterokinase (ECS) peptide DYKDDDDK (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) conjugated to KLH using maleimide. Residues of glycine and cysteine were added to the carboxy terminal end to facilitate coupling. This antibody reacts with DYKDDDDK conjugated proteins.

Isotype: IgG

Application Details

Application Notes: This antibody is optimally suited for monitoring the expression of DYKDDDDK tagged fusion proteins.  As such, this antibody can be used to identify fusion proteins containing the DYKDDDDK epitope.  The antibody recognizes the epitope tag fused to either the amino- or carboxy- termini of targeted proteins.  This antibody has been tested by ELISA and western blotting against both the immunizing peptide and DYKDDDDK containing recombinant proteins.  Although not tested, this antibody is likely functional for immunoprecipitation, immunocytochemistry, and other immunodetection techniques.  The epitope tag peptide sequence was first derived from the 11-amino-acid leader peptide of the gene-10 product from bacteriophage T7.  Now the most commonly used hydrophilic octapeptide is DYKDDDDK.  polyclonal antibody to detect DYKDDDDK conjugated proteins binds DYKDDDDK containing fusion proteins with greater affinity than the widely used monoclonal M1, M2 and M5 clones, and shows greater sensitivity in most assays.  Affinity purification of the polyclonal antibody results in very low background levels in assays and low cross-reactivity with other cellular proteins.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101368 (Western Blotting)

by: Group Muehlemann, Department of Chemistry and Biochemistry, University of Bern

No.: #101368

Date: 05/06/2017

Antigen: DYKDDDDK Tag

Lot Number: 33451

Method validated: Western Blotting

Positive Control: HeLa cells expressing DYKDDDDK-tagged THOC1 or THOC5

Negative Control: HeLa cells

Notes:

Passed. The DYKDDDDK Tag antibody ABIN99294 specifically recognizes DYKDDDDK-tagged proteins in HeLa cell extracts.

Validation Images

Increasing amounts of lysates from HeLa cells transiently expressing DYKDDDDK-tagged THOC1 were separated on an SDS-PAGE and revealed using ABIN99294 as decribed in the protocol section (lanes 3 to 7). Transiently expressed DYKDDDDK-tagged THOC5 can is also be detected using ABIN99294 (lane 1). Untransfected HeLa cell extracts were used as negative controls (lane 2).

Full Methods

Primary Antibody: ABIN99294

Secondary Antibody: rabbit anti-actin antibody (Sigma-Aldrich, A5060)

Full Protocol:

• HeLa cells are grown in DMEM/F12 W/L-GLUT (Thermo Fisher Scientific, 32500035) supplemented with 10% FCS (Amimed) and Penicillin-Strepomycin (BioConcept 4-01F00-H), at 37°C and 5% CO₂ to 70-80% confluency.
• Transfect cells with an expression plasmids encoding DYKDDDDK-tagged THOC proteins using Lipofectamine 2000 (Thermo Fisher Scientific following the manufacturer´s instructions.
• Grow cells for 48h.
• Trypsinize cells, collect and count them.
• Lyse 10⁷ cells in 1ml cold RIPA buffer (Cold Spring Harbour protocols) containing protease inhibitors (Biotool, B14003).
• Dilute the equivalent of 2x10⁵ cells (20µl) 1:2 with 2x Laemmli SDS sample buffer and heat samples up for 5min to 95°C.
• Separate the denatured samples on a denaturing freshly cast polyacrylamide gel.
• Transfer proteins onto a nitrocellulose membrane (GE Healthcare).
• Block the membrane with TBScontaining 0.5% Tween 20 and 5% milk powder for 30min at RT.
• Incubation with primary antibody
• • rabbit anti-DYKDDDDK Tag antibody (antibodies-online, ABIN99294, lot 33451) or
  • rabbit anti-actin antibody (Sigma-Aldrich, A5060)
• diluted 1:2000 in TBST 1h at RT or ON at 4°.
• Wash membrane 3x for 10min with TBST.
• Incubation with secondary antibody IRDye 800CW Goat anti-Rabbit (LiCor, 926-32211) diluted 1:10000 in TBST for 1h at RT.
• Wash membrane 3x for 10min with TBST.
• Reveal protein bands on a LI-COR Odyssey imaging system (LI-COR).

Experimental Notes:

• ABIN99294 proved a very good antibody for the detection of DYKDDDDK-tagged THOC proteins in Western blot.

• The antibody can be reused up to ten times as long as the incubation buffer contains some sodium azide.

• The antibody worked in various blocking buffers we tried containing milk or BSA and as well in Pierce WB enhancer.

Independent Validation (IF)

Validation #101369 (Immunofluorescence)

by: Group Muehlemann, Department of Chemistry and Biochemistry, University of Bern

No.: #101369

Date: 05/06/2017

Antigen: DYKDDDDK Tag

Lot Number: 33451

Method validated: Immunofluorescence

Positive Control: HeLa cells expressing DYKDDDDK-tagged EGFP or GAPDH

Negative Control: HeLa cells

Notes:

Passed. The DYKDDDDK Tag antibody ABIN99294 specifically labels DYKDDDDK-tagged proteins in HeLa cells in immunofluorescence.

Validation Images

HeLa cells transiently expressing DYKDDDDK-tagged EGFP (top row) or GAPDH (middle row) and mock-transfected HeLa cells (bottom row) were stained using DAPI (left column) or DYKDDDDK Tag antibody ABIN99294 in combination with an AF488 conjugated secondary antibody (middle column). The right column shows merged DAPI and ABIN99294 staining.

Full Methods

Primary Antibody: ABIN99294

Secondary Antibody: chicken anti-rabbit AF488 antibody conjugate (Thermo Fisher Scientific, A-21441)

Full Protocol:

• Prepare sterile 10mm cover slips (VWR, 631-1576) in empty 6-well plates.
• Seed 2x10⁵ HeLa cells expressing DYKDDDDK-tagged proteins per well (6-well plate) onto the cover slips one day prior to fixation and let them grow in DMEM/F12 W/L-GLUT (Thermo Fisher Scientific, 32500035) with 10% FCS (Amimed) and antibiotics (Penicillin-Strepomycin 4-01F00-H, Bioconcept) at 37°C and 5% CO₂ to 60-80% confluency.
• Wash cells with PBS.
• Remove culture medium and fix the cells with 1.5ml/well 4% PFA for 20-30 min at RT.
• Wash samples 3x 5min with TBS (20mM Tris-HCl, pH 7.5, 150mM NaCl).
• Incubate cells with permeabilization/blocking buffer for 30-60 min at RT.
• Incubate slides with primary rabbit anti-DYKDDDDK Tag antibody (antibodies-online, ABIN99294, lot 33451) diluted 1:200 in 100µl TBS++ (1xTBS, 0.1% Triton X100, 6% serum (or BSA 1.25gr/250mL PBS)) ON at 4°C. A negative control was incubated without primary antibody.
• Take the slides out of the fridge and leave them for 2h at RT.
• Wash slides 3x 5min with TBS++.
• Incubate slides with secondary chicken anti-rabbit AF488 antibody conjugate (Thermo Fisher Scientific, A-21441) for 1.5h at 37°C.
• Incubate slides for 30min at RT.
• Wash slides 2x with TBS.
• Counterstain with 100μL DAPI diluted to 100ng/ml in TBS for 10min at RT.
• Wash slides 3x with TBS.
• Add a drop of homemade Mowiol on the slide and mount the coverslips.
• Cover the borders with nail polish to fix the coverslips to the side.
• Take images on a Leica DMI6000 fluorescence microscope.

Experimental Notes:

Staining of transiently expressed DYKDDDDK-tagged EGFP and GAPDH with ABIN99294 resulted in the expected staining pattern.

Handling

Format: Liquid

Concentration: 1.04 mg/mL

Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2

Preservative: Sodium azide

Precaution of Use: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

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Target Details for DYKDDDDK Tag

Target: DYKDDDDK Tag

Abstract: DYKDDDDK Tag Products

Target Type: Tag

Background: Epitope tags are short peptide sequences that are easily recognized by tag-specific antibodies.  Due to their small size, epitope tags do not affect the biochemical properties of the tagged protein.  Most often, sequences encoding the epitope tag are included with the target DNA at the time of cloning to produce fusion proteins containing the epitope tag sequence.  This allows Anti epitope tag antibodies to serve as universal detection reagents for any tag-containing protein produced by recombinant means.  This means that anti-epitope tag antibodies are a useful alternative to generating specific antibodies to identify, immunoprecipitate or immunoaffinity purify a recombinant protein.  The anti-epitope tag antibody is usually functional in a variety of antibody-dependent experimental procedures.