NanoPro™ Assay: FLAG Epitope Tag on EGFP
SUMMARY
Author: Thomas Frogne PhD, Hagedorn Research Institute, Denmark (tfro@hagedorn.dk)
Primary Antibody:Anti-FLAG (Sigma, cat# F1804 and cat# F7425) and Anti-GFP (Clontech, cat# 632460)
Detection Antibody: Anti-Mouse HRP and Anti-Rabbit HRP (Cell Biosciences, p/n 040-655 and p/n 040-657)
Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The FLAG-tag
consists of a short peptide sequence (MADYKDDDDKM). EGFP was expressed with a FLAG-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.
RESULTS
FLAG-tagged EGFP expressed in mouse L-cells
Flag-tagged EGFP expressed in mouse L-cells was detected specifically by two anti-FLAG antibodies (panels A (Sigma#F7425) and B
(Sigma #F1804). Specificity was confirmed by vector only and EGFP only transfections, which did not result in any signal with the
anti-FLAG antibodies. As expected, the anti-GFP antibody (panel C (Clontech# 632460) resulted in a significant peak in the GFP only
transfection. The anti-GFP antibody recognized also as expected both Flag-tagged EGFP constructs.
PROTOCOL
Cell Preparation
Cell culture:
LTK also known as L-cells, (ATCC, cat# CRL-2648), a mouse fibroblast cell line, similar to 3T3 cells, were cultured in DMEM
containing 10% FBS and Penicillin/Streptomycin. Cells were split 1:100 every week using Trypsin.
Constructs:
Expression from EGFP vector backbone (Clontech, cat# 6084-1 and 6085-1) was driven by a CMV promoter. PCR reaction was run
from this backbone to generate plasmids expressing EGFP with either an N or C-terminal tag. All clones were verified by sequencing.
Tag Sequence:
FLAG: MADYKDDDDKM
Transfection:Cells were cultured in 6-well plates and transfected with Lipofectamine 2000, (Invitrogen, cat# 11668019), according to manufactures
instructions. The applied constructs were Empty vector, EGFP or EGFP with an N- or C-terminal FLAG tag. The transfection efficiency
was between 20% and 30%, judged by EGFP fluorescence.
Lysis buffer:
Tissue reagent I (Invitrogen, cat# FNN0071) with the addition of protease inhibitors (Roche, cat# 1 836153).
Lysis details:
Wash cells once with ice-cold PBS, aspirate well. Add 100 µL of ice-cold lysis buffer per well. Swirl around to ensure good
coverage. Incubate for 10 minutes on ice. Scrape off cells, transfer to a microfuge tube and clarify by centrifugation (17,000 x g
for 30 minutes) in a cooled centrifuge, transfer supernatant to a fresh microfuge tube.
Storage:
-80°C
Sample Preparation
Protein concentration:
0.1 mg/mL final in capillary by BCA assay
Sample diluent:
Sample Diluent (Cell Biosciences, p/n 040-649) plus 1x DMSO Inhibitor Mix
Ampholyte premix:
Premix 4-9 (nested) (Cell Biosciences, p/n 040-319)
pI Standards:
pI Standard Ladder 1 (Cell Biosciences, p/n 040-644)
Procedure:
Step 1) Dilute lysate with sample diluent to 0.2 mg/mL.
Step 2) In a separate tube, mix ampholyte premix and pI standards.
Step 3) Mix step 1 and step 2 at 1:1 to create final protein concentration.
Wash:
Wash Solution (Cell Biosciences, p/n 040-313)
Primary antibody:
Anti-FLAG (Sigma, cat# F7425 and F1804, 1:1000), anti-GFP (Clontech, cat# 632460,1:100)
in Antibody Diluent (Cell Biosciences, p/n 040-309)
Detection antibody:
Anti-Rabbit HRP (Cell Biosciences, p/n 040-656, 1:100) in Antibody Diluent
Anti-Mouse HRP (Cell Biosciences, p/n 040-655, 1:100) in Antibody Diluent
Anolyte:
Phosphoric Acid, 10 mM (Cell Biosciences, p/n 040-650)
Catholyte:
Sodium Hydroxide, 100 mM (Cell Biosciences, p/n 040-651)
Luminol/Peroxide:
Mixed 1:1 (Cell Biosciences, p/n 040-652 and p/n 040-653)
Assay Conditions
System:
NanoPro 1000
Focus conditions:
15000 μW, 40 minutes
Immobilization:
80 seconds
Wash 1:
2 x 150 seconds (default)
Primary antibody incubation:
120 minutes
Wash 2:
2 x 150 seconds (default)
Detection antibody incubation:
60 minutes
Wash 3:
2 x 150 seconds (default)
Chemiluminescence exposure:
10, 60, 120, 240, 600 and 1200 seconds