Zenon pHrodo ifl igg labeling Reagents



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For Research Use Only. Not for use in diagnostic procedures.

Zenon


 pHrodo


 iFL IgG Labeling Reagents

Catalog Nos. Z25609, Z25610, Z25611, Z25612

Pub. No. MAN0017436 

Rev. B.0

Product description

The Invitrogen

 Zenon



 pHrodo


 iFL IgG Labeling Reagents provide a fast, versatile, and reliable method to evaluate 

antibody internalization. Zenon

 labeling technology utilizes a pHrodo



 iFL Red- or pHrodo

 iFL Green-labeled Fab 



fragment (i.e., labeling reagent) directed against the Fc portion of an intact IgG primary antibody to form a labeling 

complex. Formation of the Fab–antibody complex occurs in less than 5 minutes (Figure 1). Because the pHrodo

 iFL 


dyes dramatically increase in fluorescence as the pH of their surroundings becomes more acidic, the antibodies coupled 

with the Zenon

 pHrodo


 iFL IgG Labeling Reagents provide an excellent measure of endocytic activity based on the 

acidification of the labeled antibodies as they are ingested by the cell (Figure 2). 

The Zenon

 pHrodo


 iFL IgG Labeling Reagents contain sufficient material to label one to four 96-well plates, depending 

on the Zenon

 labeling reagent concentration used. 



Note: pHrodo

 iFL dyes are extremely sensitive to their local environment; therefore, the pH response in your system 



needs to be determined empirically.

Table 1 Contents and storage

Product

Cat. No.


Amount*

Storage**

Zenon



 pHrodo



 iFL Green Mouse IgG Labeling Reagent

Z25609

250 μL


• 2–6°C

• DO NOT FREEZE

• Protect from light

Zenon


 pHrodo


 iFL Red Mouse IgG Labeling Reagent

Z25610

Zenon


 pHrodo


 iFL Green Human IgG Labeling Reagent

Z25611

Zenon


 pHrodo


 iFL Red Human IgG Labeling Reagent

Z25612

* 300 µg Fab fragment/mL in 0.1 M sodium phosphate, 0.1 M NaCl, pH 7.5, containing 5 mM sodium azide



** When stored as directed the product is stable for at least 6 months.

Approximate fluorescence excitation and emission maxima: pHrodo

 iFL Green: 505/530 nm; pHrodo



 iFL Red: 560/585 nm.

USER GUIDE



Zenon

 pHrodo



 iFL IgG Labeling Reagents

 | 2

Figure 2. Intact IgG primary antibodies labeled with the Zenon



 pHrodo


 iFL IgG Labeling Reagents 

show dramatic increases in fluorescence as they are internalized by the cell and the pH of their 

surroundings become more acidic.

Materials required but  

not provided

•  Whole IgG primary antibody

•  Suspension cells at 2 × 10

6

 cells/mL in cell culture medium or adherent cells in a 



96-well plate at 5,000–10,000 cells/well in cell culture medium

•  Cell culture medium

•  96-well plates 

•  Instruments to analyze cells probed with Zenon

 pHrodo


 iFL-labeled IgG (flow 

cytometer for suspension cells, or fluorescence microscope or high-content analysis 

instrument for adherent cells)

Figure 1. The Zenon

 labeling scheme. An unlabeled IgG is incubated with the Zenon



 pHrodo


 iFL 


IgG Labeling Reagent, which contains a fluorophore-labeled Fab fragment. The labeled Fab fragment 

binds to the Fc portion of the IgG antibody.




Zenon

 pHrodo



 iFL IgG Labeling Reagents

 | 3

Procedural guidelines



•  Zenon

 pHrodo



 iFL IgG Labeling Reagents are goat Fab fragments selective for 

the Fc portion of human or mouse IgG antibodies. They are used to non-covalently 

couple the pHrodo

 iFL Red- or pHrodo



 iFL Green-labeled Fab fragments to the 

unconjugated human or mouse IgG antibodies in 5 minutes, leaving the antigen 

binding site of the antibody unmodified, while providing a consistent degree of 

labeling (DOL) of 3 to 5 Fab molecules per IgG (Figure 1).

•  The labeled Fab fragments (i.e., labeling reagents) have been affinity purified during 

their preparation to ensure their high affinity and selectivity for the Fc portion of 

the primary antibody. Because this labeling is based on immunoselectivity, the 

Zenon



 labeling method does not require the removal of exogenous proteins such as 



serum albumin or amine-containing buffers from the antibody prior to forming the 

complex. Cross-reactivity is low with antibodies from other species.

•  Formation of the Fab–antibody complex (i.e., labeling complex) occurs in less than 

5 minutes, and nearly all of the primary antibodies in the mixture are labeled. 

Complexes formed using this technology display fluorescence intensity similar to 

that of directly labeled primary antibodies. 

•  The extent of antibody labeling (and thus the fluorescence intensity of the probe) can 

be adjusted by varying the amount of Zenon

 labeling reagent that is added, i.e. by 



varying the molar ratio of labeled Fab fragment to primary antibody.

•  The protocol described here is for performing internalization assays with one 96-well 

plate of antibodies, each at 40 nM (6 µg/mL) with Zenon

 pHrodo



 iFL IgG labeling 

reagent at 120 nM (6 µg/mL). This molar ratio is a suggested starting point and 

represents the minimum ratio for adequate signal in most applications. Experiments 

with antibodies against highly-expressed or rapidly internalizing antigens may 

obtain satisfactory signal with lower antibody concentrations.  

 - One 96-well plates at 40 nM antibody/120 nM Zenon

 labeling reagent



 - Two 96-well plates at 20 nM antibody/60 nM Zenon

 labeling reagent



 - Four 96-well plates at 10 nM antibody/30 nM Zenon

 labeling reagent



•  For larger or smaller quantities of antibody, the amounts of the reagents specified in 

the protocol can be scaled accordingly. The Zenon

 IgG labeling reaction does not 



require the removal of bovine serum albumin (BSA) or other stabilizing proteins that 

may be present in antibody preparations. Antibodies contained within serum may 

also be directly labeled and do not require purification of the antibody either prior to 

or after labeling.

•  The pHrodo

 iFL Green dye has excitation and emission maxima of approximately 



505 nm and 530 nm, respectively, and can be detected with standard FITC 

(fluorescein) or Alexa Fluor

 488 filters. The pHrodo



 iFL Red dye has excitation and 

emission maxima of approximately 560 nm and 585 nm, and can be detected with 

standard TRITC (tetramethylrhodamine) or Alexa Fluor

 555 filters.




Zenon

 pHrodo



 iFL IgG Labeling Reagents

 | 4

Methods


Prepare 4X antibody working 

solution


 1.1 

Prepare sufficient volume of 4X working solution of antibody in cell culture medium so 

that you can use 25 µL for each sample. For example, to fill one 96-well plate, prepare 

2.5 mL of working antibody solution. 

Note: 40 nM is a good starting concentration for many antibodies, so a 4X stock will be 

160 nM.  



 1.2 

Aliquot 25 µL of 4X antibody working solution to each well of a 96-well plate.

Prepare 4X Zenon

 working 



solution

 

2.1  

Prepare 4X working solution of Zenon

 pHrodo


 iFL IgG Labeling Reagent. For 

example, for one 96-well plate, add 200 µL of Zenon

 pHrodo



 iFL IgG labeling reagent 

to 2.3 mL of cell culture medium to prepare 2.5 mL of Zenon

 working solution.



 

2.2  

Aliquot 25 µL of 4X Zenon

 working solution to each well of the 96-well plate from 



Step 1.2. Incubate for 5 minutes at room temperature to allow the labeling complexes to 

form.


Label suspension cells

 3.1 

Prepare at least 5 mL of suspension cells at 2 × 10

6

 cells/mL in cell culture medium. 



 

3.2

  Add 50 µL of cells to each well of the 96-well plate containing the antibody and the 

Zenon



 labeling reagent (from Step 2.2). 



 

3.3

  Incubate the cells with the labeling complex for 1–24 hours under standard cell culture 

conditions. Add other antibodies or cell labels as desired. 

 

3.4

  Analyze cells using flow cytometry.

Label adherent cells

 

4.1

  Prepare a 96-well plate containing 5,000–10,000 cells/well. After the cells adhere, adjust 

volume so that each well contains 50 µL of culture medium. 

 

4.2

  Add 50 µL of the labeling complex (from Step 2.2) to each well of the 96-well plate.

 

4.3

  Incubate the cells with the labeling complex for 1–24 hours under standard cell culture 

conditions. Add other antibodies or cell labels as desired. 

 

4.4

  Analyze cells using fluorescence microscopy or high content analysis.




Purchaser notification

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Visit thermofisher.com/support for the latest in services and support, including:

•  Worldwide contact telephone numbers

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- Certificates of Analysis

 

- Safety Data Sheets (SDSs; also known as MSDSs)



Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.

Limited Product Warranty 

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life 

Technologies’ website at 

www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at 

thermofisher.com/support.

The information in this guide is subject to change without notice.

For Research Use Only. Not for use in diagnostic procedures.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, 

INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0017436

Revision


Date

Description

B.0

26 January 2018



Remove definition of labeling from Introduction.

A.0


06 November 2017

New document

Important Licensing Information 

These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited 

Use Label Licenses.

 

 Manufacturer: Life Technologies Corporation | 29851 Willow Creek Road | Eugene, OR 97402



All trademarks are the property of Thermo Fisher Scientific and its subsidiaries, unless otherwise specified.

©2018 Thermo Fisher Scientific Inc. All rights reserved. 

26 January 2018

Ordering information

Cat. No. 

Product name . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Unit size

Z25609 Zenon

 pHrodo



 iFL Green Mouse IgG Labeling Reagent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 μL

Z25610 Zenon

 pHrodo



 iFL Red Mouse IgG Labeling Reagent  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 μL

Z25611 Zenon

 pHrodo



 iFL Green Human IgG Labeling Reagent  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 μL

Z25612 Zenon

 pHrodo



 iFL Red Human IgG Labeling Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 μL




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