Anti-Human RAGE Therapeutic Antibody scFv Fragment (XT-M4)

Published Data
Datasheet
MSDS
COA

SPR

Figure 1 3B4 has a high affinity to mouse RAGE while the control M4 does not bind mouse RAGE.

SPR sensorgrams of A. anti-RAGE scFv (3B4) and B. control scFv (M4) using the Langmuir 1:1 binding model.

Kim, H. Y., Wang, X., Kang, R., Tang, D., Boone, B. A., Zeh III, H. J., ... & Edwards, W. B. (2018). RAGE-specific single chain Fv for PET imaging of pancreatic cancer. PloS one, 13(3), e0192821.

Figure 2 Confocal microscopic images of RAGE expressing Panc02 cells.

Live cells were incubated with 3B4-Cy5 and M4-Cy5. After fixation, the nucleus was counterstained with DAPI (Scale bar = 10 μm).

Kim, H. Y., Wang, X., Kang, R., Tang, D., Boone, B. A., Zeh III, H. J., ... & Edwards, W. B. (2018). RAGE-specific single chain Fv for PET imaging of pancreatic cancer. PloS one, 13(3), e0192821.

Figure 3 Flow cytometric analysis with fixed and live Panc02 cells demonstrate antibody-binding.

Cells were stained with 3B4-Cy5 and M4-Cy5 (n ≥ 3, ± SEM, *** p < 0.005 to the untreated cells).

Kim, H. Y., Wang, X., Kang, R., Tang, D., Boone, B. A., Zeh III, H. J., ... & Edwards, W. B. (2018). RAGE-specific single chain Fv for PET imaging of pancreatic cancer. PloS one, 13(3), e0192821.

Datasheet

MSDS

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Specifications

Applications

Application Notes
The RAGE antibody has been reported in applications of Surface Plasmon Resonance, Immunofluorescence, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Neutralization.
For Surface Plasmon Resonanc: A Biacore X100 was used for binding kinetic study of purified scFvs. To evaluate the binding affinity of scFvs, recombinant mouse RAGE Fc Chimera was immobilized on a CM5 sensor chip via an amine coupling (~2,000 RU). The carboxyl groups of the reference cell were activated and quenched with aminoethanol. The anti-RAGE-Mab, scFvs and conjugates were diluted in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4) and flowed over the surface for 180 s at a rate of 30 μL/min followed by dissociation for 600 seconds. After each sample injection, the surface was regenerated with consecutive injections of 5 μL of 0.3% SDS solution and 3.3 μL 50 mM NaOH. All sensorgrams were double referenced by subtracting the surface effect from the control flow cell and the buffer effect from the blank buffer. The calculated kinetic values ka, kd, and KD were obtained using Biacore X100 Evaluation Software assuming the Langmuir 1:1 binding model. The fitted curves are superimposed on the binding isotherms.
For Flow Cytometry: Panc02 cells were harvested by trypsinization and fixed with 4% PFA. 5 x 104 cells were used for each labeling condition (n ≥ 3). Cells were incubated with 3B4-Cy5 and M4-Cy5 (2 μg) in the incubation buffer (100 μL) for 30 min on ice. Anti-mouse RAGE Mab (1/100) and Alexa Fluor 488 conjugated anti-mouse antibody (1/200) were sequentially incubated with cells for 30 min on ice. After each incubation, cells were washed with cold PBS three times. The same labeling conditions were used for live cell staining. Flow cytometric analysis was performed on a FACS LSR Fortessa flow cytometer using FACSDiva software provided by the University of Pittsburgh Cancer Institute Flow and Imaging Cytometry core facility, and the data was analyzed using VenturiOne software.

Target

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3D IHC3D Immunohistochemistry

ActivActivation

AgonistAgonist

ApopApoptosis

BABioassay

BIBioimaging

BlockBlocking

Cell ScreeningCell Screening

SeparationCell Separation

ChIPChromatin Immunoprecipitation

CMCDComplement Mediated Cell Depletion

CostimCostimulation

CytCytotoxicity

DepletionDepletion

DBDot Blot

EMElectron Microscopy

ELISAEnzyme-linked Immunosorbent Assay

ELISPOTEnzyme-linked Immunosorbent Spot

FCFlow Cytometry

FuncSFunctional Assay

GSGel Super Shift Assay

HAHemagglutination

IAImmunoassay

IBImmunoblotting

ICCImmunocytochemistry

IDImmunodiffusion

IFImmunofluorescence

IHCImmunohistochemistry

IHC-FrImmunohistochemistry-Frozen

IHC-PImmunohistochemistry-Paraffin

REImmunohistology - Resin Sections

IPImmunoprecipitation

IRMAImmunoradiometric Assay

SHIn situ hybridization

InhibInhibition

ICFCIntracellular Staining for Flow Cytometry

KO/KD-WBKnockout/Knockdown target confirmation by Western Blot

Live cell imagingLive cell imaging

CyTOF®Mass Cytometry

MeDIPMethylated DNA Immunoprecipitation

MultiplexMultiplex bead-based assay

NeutNeutralization

PPProtein Purification

PGProteogenomics

RIRadial Immunodiffusion

RIARadioimmunoassay

StimStimulation

SPRSurface Plasmon Resonance

TCTissue Culture

TBTurbidimetry

WBWestern Blot

Anti-Human RAGE Therapeutic Antibody scFv Fragment (XT-M4)