Manual of GFP IP Kit
Cat. No. GFP-IP
Catalog No.

GFP-IP-50

GFP-IP-200

Size50 rxns

200 rxns

Price (USD)

$250

$920

250ul/$25.00
order separately

4x250ul/$90.00
order separately

Biotinylated control IgY(0.2mg/ml)
Introduction

This kit is for capturing GFP and its fusion proteins from cell lysate. The combination of biotinylated
chicken IgY against GFP protein as IP antibody and streptavidin conjugated magnetic beads as isolating
matrix confers the kit with several advantages:1) The chicken IgY anti GFP antibody in this kit is antigen affinity purified, which will greatly reduce theuse of the beads, meanwhile minimize the non-specificity, as compare to using total IgG or total IgY in IP.
2) Because of the extremely high affinity of streptavidin to biotin, the streptavidin-magnetic beads cancapture biotinylated IgY GFP/GFP complex at a very high efficiency, which will increase the sensitivity ofcapture. This kit will allow users to capture as low as 1ng GFP protein and as high as 330ng GFP protein.
3) The Fc region of chicken IgY is very different from Fc region of mammalian IgG. When protein
detecting antibodies from mammalian species are used in Western Blot after the immunoprecipitation,
it will not show undesired bands since anti-IgG secondary antibodies do not bind or bind very little to IgY.
Kit contents
Catalog No.

GFP-IP-50

GFP-IP-200Size

50 rxns

200 rxns

Biotinylated IgY anti-GFP (0.2mg/ml)

260ul

1020ulStreptavidin-Magbeads (10mg/ml)

65ul

250ul

Eluting solution

1.25ml5ml

Neutralization solution

0.2ml

0.8ml

Reminding1.
Users must use proper ways to prepare cell lysate to assure 1) chromosomal DNA is
disrupted so that the lysate is not glutinous, and 2) proteolysis in the lysate isinhibited.

2.
3.

This capture protocol is developed using round bottom 96-well plate, users may choose
other formats, such as microcentrifuge tubes or PCR tubes.
For short period storage (up to 2 weeks), store the whole kit at 4oC; for long term
storage, take out the tube labeled Biotinylated IgY anti GFP and store at -20oC, andstore the rest at 4oC. Avoid frequent frozen-thaw cycles.

Protocol1. Pretreat Streptavidin Magbeads
Fully suspend beads by vortex and take desired volume of the beads suspension (12ug for eachcapturing reaction). Remove the liquid on magnetic separator (provided by user), and wash thebeads three times with PBST (0.05% Tween 20), 10 volumes of beads suspension each time,using magnetic separator. Resuspend the beads in PBST to make 2mg/ml. It is ready to use.
2. To 100-200ul clear cell lysate, add 5ul (0.2ug/ul) Biotinylated IgY anti-GFP, mix well.
3. Incubate at room temperature for 1 hour with shaking.
4. Add 6 ul (2ug/ul) preteated streptavidin Magbeads (fully suspended) to the mixture, mix well.
5. Incubate at room temperature for 30 minutes or 4oC for overnight with shaking. During theincubation, pipette the mixture couple times in every 10 minutes. (if tube is used, theincubation can be performed on end-to-end rotator or other appropriate equipment, then noneed for interval pipetting)
6. Isolate the beads from solution on magnetic separator. (you may save the liquid)
7. Wash the beads with 200ul PBST (0.05% Tween 20) three times, by using magnetic separator.
8. After wash, add 10 or 20ul Eluting solution, pipette to completely suspend the beads. Then
separate the solution from the beads on magnetic separator. Transfer the elutant to a new tube
that has been pre-added with 1 or 2ul Neutralization solution. (with this eluting condition, no orvery little IP antibody will be eluted, which greatly reduces interference from the IP antibody
bands in Western Blot where the 1st antibody used is from the same species as the IP antibody.)
The elutant is now ready for downstream analysis.
ExampleWestern Blot analysis of the capture efficiency
1: 1ng GFP input
2: 1ng GFP IP captured in 100ul tissue lysate
3: 10ng GFP input
4: 10ng GFP IP captured in 100ul tissue lysate
1st antibody: IgY anti-GFP

GFP

1234