chromotek gfp-multitrap

8/4/2019 ChromoTek GFP-multiTrap

http://slidepdf.com/reader/full/chromotek-gfp-multitrap 1/3

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®.

A

B




Protocols

1. Immunoprecipitation

•For one immunoprecipitation reaction resuspend cell pel-

let (~107 cells) in 200 μL lysis buffer by pipetting (or using

a syringe)

•Place the tube on ice for 30 min with extensively pipetting

every 10 min•Spin cell lysate at 20.000x g for 5-10 min at 4°C

•Transfer supernatant to a precooled tube. Adjust volume

with dilution buffer to 500 μL-1000 μL. Discard pellet.

The cell lysate can be frozen at this point for long-term stor-

age at minus 80°C.

For immunoblot analysis dilute 50 μL cell lysate with 50 μL

4x SDS-sample buffer (→refer as input)

•Add 50 μL (half area GBP-Plate) or 100 μL (full area GBP

plate) cell lysate per well

•Incubate for 2 hours at 4°C under shaking (800rpm)

For western blot analysis dilute 50 μL cell lysate with 50 μL4x SDS-sample buffer (→refer as ow-through)

•Discard remaining supernatant

•Wash wells two times with 100 μL- 200 μL wash buffer

•Add 100 μL dilution buffer for Fluorescence intensity mea-

surements

Add 10 μL of elution buffer to wells and transfer it to a tube.

Buffer with 1M Tris pH 7.5 to an end concentration of 100

mM Tris and dilute with 10 μL 4x SDS-sample buffer (→refer

as bound)

2. In vitro binding assays after IP

2.1. In vitro histone-tail peptide binding assay

•After one-step purication of GFP fusion proteins with

the 96-well GBP plate in half-area plates (1), the wells are

blocked with 100 μL blocking buffer for 30 minutes at 4°C

under shaking (800rpm)

•Discard blocking solution

•Equilibrated wells with 50 μL dilution buffer supplemented

with 0.05% Tween

•Add peptides to a nal concentration of 0.15 μM

•Incubate at room temperature for 20 min under shaking

(800rpm)

•Discard supernatant

•Wash wells two times with 100 μL wash buffer


surements

2.2. In vitro DNA binding assay

•After one-step purication of GFP fusion proteins with the

96-well GBP plate in full-area plates(1), the wells are equilibrated with 100 μL dilution buffer sup-

plemented with 2mM DTT and

100ng/μL BSA

•Add uorescent-labeled DNA probe to a nal concentration

of 0.15 μM

•Incubate at room temperature for 30 min under shaking

(800rpm)

•Discard supernatant

•Wash wells two times with 100 μL dilution buffer


surements

2.3. In Vitro Protein-Protein binding assay

•After one-step purication of GFP fusion proteins with the

96-well GBP plate in full-area plates

(1), the wells are equilibrated with 100 μL dilution buffer sup-

plemented with 2mM DTT and

100ng/μL BSA

•Prepare cell lysate of RFP-fusion protein as described for

GFP-fusion proteins

•Add 50 μL (half area GBP-Plate) or 100 μL (full area GBP

plate) cell lysate per well•Incubate for 30 minutes at 4°C under shaking (800rpm) For

western blot analysis dilute 50 μL cell lysate with 50 μL 4x

SDS-sample buffer (→refer as ow-through)

•Discard remainning supernatant

•Wash wells two times with 100 μL- 200 μL wash buffer


surements. Add 10 μL of elution buffer to wells and transfer it

to a tube. Buffer with 1M Tris pH 7.5 to an end concentration

of 100 mM Tris and dilute with 10 μL 4x SDS-sample buffer

(→refer as bound)

Box 1 | Product List

GFP-multiTrap® Plates

Chromotek-GFP-multiTrap® ABP-CM-GMULT1 1 Plate

ABP-CM-GMULT5 5 Plates




ProtocolsContinued

3. Fluorescence Measurements

3.1. Quantication with Tecan Innite M1000 plate reader

•GFP: : 490±10 nm and 511±10 nm

•RFP: 586±5 nm and 608±10 nm

•TAMRA: 560±5 nm and 586±5 nm

•DNA: according to label

For quantication, uorescence intensity measurements are

adjusted using standard curves from labelled probes with

known concentrations.

Suggested Buffers

Lysis buffer

20 mM Tris/HCl pH7.5

150 mM NaCl

0.5 mM EDTA

0.5% NP-40

Optional:

1 mM PMSF has to be freshly added

1x Protease Inhibitor Cocktail has to be freshly added

For nuclear/chromatin proteins:

DNase nal conc. 1 μg/μl

2.5 mM MgCl2

Dilution buffer


150 mM NaCl

0.5 mM EDTA

Wash buffer


50-500 mM NaCl

0.5 mM EDTA

Blocking Buffer

3% milk solved in TBS-T (0.075% Tween)

Elution buffer (freshly prepared)

300 mM Glycin pH2.5

Box 3 | GFP-multiTrap Overview

Welcome to Join in the Discussion at Allele’s Network:

Allele News: news.allelebiotech.com

Allele Blog: blogs.allelebiotech.com

Allele Facebook: http://www.facebook.com/…

Allele Twitter: http://www.twitter.com/allele_biotech

Allele Myspace: http://www.myspace.com/allelebiotech

chromotek gfp-multitrap

Documents