Allele Biotech-Introducing Cost Effectiveness to Research G reen uorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. However, among the most commonly used tags for immuno- precipitation (a brief review in Box 1), the use of GFP is limited due to the previously available anti-GFP antibod- ies, either polyclonal or monoclonal, not being comparable to those against other tags. GFP-multiTrap® is a high qual- ity GFP-bingding protein based on a single domain antibody immobilized in wells. It is characterized by a small barrel shaped structure (13 KDa, 2.5 nm X 4.5 nm) and a very high stability (stable up to 70°C, functional within 2 M NaCl or 0.5% SDS). From detailed in vitro binding analysis, we deter- mined that one molecule GFP-Trap® binds one molecule GFP in a stable stoichiometric complex. The dissocia- tion constant (Kd) lies with 0.59 nm within the picomolar range compara- ble to conventional antibodies. GFP-multiTrap® is available in black 96-well plate format with clear bottom for colorimetric, chemiluminescence and uorescence detection methods. With much greater stability, specicity, and afnity, GFP-Trap®, the recent addition to antibodies for immunopre- cipitation should make GFP in line to become the most suitable tags for im- munoprecipitation assays. Datas from direct comparison of the GFP-Trap® with conventional antibodies will be shown in Box 2. Besides wtGFP, GFP-multiTrap® can also bind to eGFP and GFPS65T as well as to YFP and eYFP. It recog- nizes and binds a three dimensional epitope at the beta barrel structure. Interestingly it does not bind to CFP, which is due to the fact, that CFP ex- hibit an amino acid exchange within the recognized epitope. In addition we could not detect any binding to red uorescent proteins derived from DsRed (RFP-Trap® is available as another product line). Meanwhile, as GFP-Trap® recognizes the beta barrel structuure of GFP, it does not recognise unfolded or denatured GFP (e.g. on immunoblots). Chromotek-GFP-multiTrap ® , GFP-Trap® immobilized in wells to test your GFP fusion proteins for pep- tide, protein, DNA or RNA binding. Box 1 | Tags for Immunoprecipitation T o achieve effective immunoprecipitation, a researcher must rst overcome the difculty of nding usable antibodies against a target of interest. Using tags that are fused to the C- or N-terminus of the targe t protein is common practice. In general, while keeping mindful of the unique nuances with each biological system, choosing tags that have been tested in many situations and been proven to be non-interfering is ideal. The most commonly used tags are: FLAG, Myc, HA, V5, T7, and His, which are quite small in size and in theory less likely to interfere. GST and GFP are well documented to form self-contained and stable structures independent of their fusion partners and proven to not interfere in many cases despite their larger size (in be- tween 20-30kD). A top choice for pulldown experiments, GST c an bind to glutathione beads directly. GFP and variants are excellent tags having both the advantages ofbeing a visualization module to follow the protein both inside cells and during pull- down. However its use is limited due to the previously available anti-GFP antibodies, either polyclonal or monoclonal, not being comparable to those against other tags. With much greater stability, specicity, and afnity, GFP-Trap®, the recent addition to antibodies for immunoprecipitation should make GFP in line to become the most suitable tags for immunoprecipitation assays. Box 2 | Comparative Immunoprecipitation Assay [A] GFP-multiTrap® Immunoprecipitations (IP) of GFP and GFP-fusion proteins (CBX1) from ex- tracts of GFP-producing human cells. Input (I), non-bound (FT), and bound (B) fractions were sepa- rated by SDS-PAGE. [B] Fraction input percentages of GFP and GFP-CBX1 and their GFP brightness displaying the efciency of pulldown using the GFP-multiTrap®. A B
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Allele Biotech-Introducing Cost Effectiveness to Research
Green uorescent proteins (GFP)and variants thereof are widely
used to study protein localization and
dynamics. However, among the most
commonly used tags for immuno-
precipitation (a brief review in Box
1), the use of GFP is limited due to the
previously available anti-GFP antibod-
ies, either polyclonal or monoclonal,
not being comparable to those against
other tags.
GFP-multiTrap® is a high qual-
ity GFP-bingding protein based on a
single domain antibody immobilizedin wells. It is characterized by a small
barrel shaped structure (13 KDa, 2.5
nm X 4.5 nm) and a very high stability
(stable up to 70°C, functional within 2
M NaCl or 0.5% SDS). From detailed
in vitro binding analysis, we deter-
mined that one molecule GFP-Trap®
binds one molecule GFP in a stable
stoichiometric complex. The dissocia-
tion constant (Kd) lies with 0.59 nm
within the picomolar range compara-
ble to conventional antibodies.
GFP-multiTrap® is available in black
96-well plate format with clear bottom
for colorimetric, chemiluminescence
and uorescence detection methods.
With much greater stability, specicity,
and afnity, GFP-Trap®, the recent
addition to antibodies for immunopre-
cipitation should make GFP in line to
become the most suitable tags for im-munoprecipitation assays. Datas from
direct comparison of the GFP-Trap®
with conventional antibodies will be
shown in Box 2.
Besides wtGFP, GFP-multiTrap® can
also bind to eGFP and GFPS65T as
well as to YFP and eYFP. It recog-
nizes and binds a three dimensionalepitope at the beta barrel structure.
Interestingly it does not bind to CFP,
which is due to the fact, that CFP ex-
hibit an amino acid exchange within
the recognized epitope. In addition
we could not detect any binding to
red uorescent proteins derived from
DsRed (RFP-Trap® is available as
another product line). Meanwhile,
as GFP-Trap® recognizes the beta
barrel structuure of GFP, it does not
recognise unfolded or denatured GFP
(e.g. on immunoblots).
Chromotek-GFP-multiTrap®, GFP-Trap®
immobilized in wells to test your GFP fusion proteins for pep-tide, protein, DNA or RNA binding.
Box 1 | Tags for Immunoprecipitation
To achieve effective immunoprecipitation, a researcher must rst overcome the
difculty of nding usable antibodies against a target of interest. Using tags that are
fused to the C- or N-terminus of the target protein is common practice. In general,
while keeping mindful of the unique nuances with each biological system, choosing
tags that have been tested in many situations and been proven to be non-interfering
is ideal. The most commonly used tags are: FLAG, Myc, HA, V5, T7, and His, which
are quite small in size and in theory less likely to interfere. GST and GFP are well
documented to form self-contained and stable structures independent of their fusion
partners and proven to not interfere in many cases despite their larger size (in be-
tween 20-30kD). A top choice for pulldown experiments, GST can bind to glutathione
beads directly. GFP and variants are excellent tags having both the advantages of
being a visualization module to follow the protein both inside cells and during pull-down. However its use is limited due to the previously available anti-GFP antibodies,
either polyclonal or monoclonal, not being comparable to those against other tags.
With much greater stability, specicity, and afnity, GFP-Trap®, the recent addition
to antibodies for immunoprecipitation should make GFP in line to become the most
suitable tags for immunoprecipitation assays.
Box 2 | Comparative Immunoprecipitation Assay
[A] GFP-multiTrap® Immunoprecipitations (IP) of GFP and GFP-fusion proteins (CBX1) from ex-tracts of GFP-producing human cells. Input (I), non-bound (FT), and bound (B) fractions were sepa-rated by SDS-PAGE.
[B] Fraction input percentages of GFP and GFP-CBX1 and their GFP brightness displaying theefciency of pulldown using the GFP-multiTrap®.