A new method to address cross-reactivity of antibodies

Slides:



Advertisements
Similar presentations
Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry
Advertisements

Exploration of Phylogeny of Serum Proteins Using the Western Press-Blot Procedure.
Reference Schaechter chapters 45 and 55 Schaechter chapter 32 for Polio vaccines. There is a table that compares live and killed vaccines. Brooks chapter.
Microarrays: Tools for Proteomics
MCB 730: Southern Blot Lab Southern, Northern & Western blotting Flowchart Detailed Protocol.
Enzyme-linked Immunosorbent Assay
Complement based techniques Complex protein system by which certain antibodies are capable of killing cells Proteins of the complex system are thermolabile.
Protein arrays LEAPS technology An array of 110 different antibodies incubated with various levels of the fluorescently labelled cognate antigens in a.
Enzyme-Linked Immunosorbent Assay ELISA 1Dr. Nikhat Siddiq.
Lab#6 Western Blotting BCH 462[practical].
Western Blotting.
Finish up array applications Move on to proteomics Protein microarrays.
Protein Lysate Microarrays Clay Scott Ryan McConnell Shannon Neeley.
Western blotting. Antibodies in the Immune System Structure: 2 heavy chains + 2 light chains Disulfide bonds 2 antigen binding sites Isotypes: IgG, IgM,
Simple ELISA protocol 1. Coat antigen onto microplate
 YEOH HUI SHIH –INTRODUCTION TO HIV  RAJAMANI- ELISA  TIEN WEI PING – WESTERN BLOTTING  YEO HUI YUN –OVERVIEW & CONCLUSION.
Monoclonal Antibody Production Against Synthetic Peptides Representing PrP c and Recombinant Prion Proteins (rPrP) By Kaitlin McDaniel, Dept. of Molecular.
Page 1 LysatesLysates in Creative BioMart — Creative BioMart.
Lab# 5 Western Blot BCH 462[practical].
Immunoassays are tests that take advantage of the specificity of antibodies to their protein epitotes. Qualitative example: pregnancy test kits, HIV.
Biotechniques (BIOL 410) Immunoblotting.
SDS-Polyacrylamide Gel Electrophoresis Agarose vs. SDS-PAGE
Blot, Blot, Western Baby Kristin B. Dupre June 30th, 2011.
2D PAGE/Western blotting analysis for the identification of possible post-translation modification of Hsp27 from the spinal cord tissue of the SCI rat.
The RD1-encoded antigen Rv3872 of Mycobacterium tuberculosis as a potential candidate for serodiagnosis of tuberculosis  P. Mukherjee, M. Dutta, P. Datta,
Distribution of phosphorylation sites identified in the cytosolic phosphoproteome.A, numbers of approved phosphopeptides, previously phosphorylated peptides,
SDS-PAGE and immunoblot analysis of F4:5 progeny of a 10/81b × PI cross. SDS-PAGE and immunoblot analysis of F4:5 progeny of a 10/81b × PI
Cellular localization of the chimeric Iff5-Iff1C, Iff5-Iff2C, Iff5-Iff4C, Iff5-Iff7C, and Iff5-Iff10C proteins. Cellular localization of the chimeric Iff5-Iff1C,
Differential conjugation of endogenous SUMO-1 and endogenous SUMO-2/3 to target proteins.A and B, SUMO-1 and SUMO-2 proteins were produced in E coli and.
SDS-PAGE analysis of mature seed from lines Patriot and PI
Frequency distribution of the GRAVY of the theoretical proteins (open bars) and of 110 genes encoding proteins identified on a 2-D electrophoresis gel,
Two-dimensional electrophoresis results and validation with Western blotting. Two-dimensional electrophoresis results and validation with Western blotting.
Schematic representation of proteogenomic annotation strategy.
Two-dimensional gel silver staining and two-dimensional immunoblotting using antibody to 3-nitrotyrosine. Two-dimensional gel silver staining and two-dimensional.
Experimental setup.A, topology models of the overexpressed membrane protein GFP fusions. Experimental setup.A, topology models of the overexpressed membrane.
Success rates in validation of antibodies from external providers
Technical reproducibility and biological variability.
Characterization of aggregates isolated from E
Protein microarrays for validation of antibodies.
A, Western blot analysis of fetuin-A in AGA (lanes 1–5 and 10–13) and IUGR (lanes 6–9, 14, and 15) UC plasma samples. A, Western blot analysis of fetuin-A.
Testing the effectiveness of the three-step peptide fractionation method.A, μLC mass chromatograms of SCX fractions for an acidic FFE fraction. Testing.
Analysis of whole cell lysates by Western blotting and 2D gel electrophoresis.A, cells overexpressing GFP fusion proteins and control cells were cultured.
Identification of chaperonin GroEL (Rv0440) with representative MS/MS spectrum. Identification of chaperonin GroEL (Rv0440) with representative MS/MS spectrum.A,
Comparison of mapped epitopes and peptides identified in immuno-SILAC screening of polyclonal antibodies against trypsin-digested PrESTs. Comparison of.
Western blot analysis of fetuin-A in AGA and IUGR UC plasma after digestion with glycosidases showing the elimination of the IUGR-related isoforms following.
Chromatograms, MS and MS/MS spectra obtained by LC-MS/MS (Q-TOF) of peptides from UPIII identified after Western blotting followed by on-membrane digestion.
Cellular localization of the Iff2, Iff3 (or -9), Iff5, Iff6, and Iff8 proteins. Cellular localization of the Iff2, Iff3 (or -9), Iff5, Iff6, and Iff8 proteins.
Localization of the Iff8 extended protein.
Protein kinase fractionation and enrichment by multicolumn affinity chromatography of lysate from A549 cells. Protein kinase fractionation and enrichment.
Immunoblot analysis of apoptotic HCT-8 cell extracts separated by SDS-PAGE. Immunoblot analysis of apoptotic HCT-8 cell extracts separated by SDS-PAGE.
Antibody specificity ascertained by 2D-PAGE Western immunoblotting (IEF) of total cellular protein extracts from the RT4 human bladder cancer cell line.
Western blotting analysis of purified cytoplasmic membranes.
IgG Autoantibodies from Bullous Pemphigoid (BP) Patients Bind Antigenic Sites on Both the Extracellular and the Intracellular Domains of the BP Antigen.
Figure S3. Zhang et al. A B Myc-SIRT1 - + sir2a+/+ sir2a-/-
PARP cleavage is detected in TA cells, but not in KSC
TP53 western blot of primary cultured tail cells from both wild-type (WT) and Tp53Δ11/Δ11 (−/−) rats. TP53 western blot of primary cultured tail cells.
Caspases are activated earlier in young TA (yTA) than in KSC
Western blot analysis of histone H1.X.
Systematic viral epitope scanning (VirScan).
p65 depletion increases basal protein aggregation and insolubilization
4E-BP is present in an 80 kDa complex in unfertilized eggs.
Recognition of naturally processed HTLV-1 Tax protein derived from HAM patient's PBMC. A, HTLV-1 Tax protein content was evaluated by Western blot analysis.
Mapping the Pirh2 and p73 interaction sites.
Western immunoblot of Acanthamoeba whole-cell lysates reacted with normal human serum, demonstrating immunoreactivity against Acanthamoeba antigens. Western.
Effect of other nucleoside analogues on p38 MAPK phosphorylation levels. Effect of other nucleoside analogues on p38 MAPK phosphorylation levels. A, MM.1S.
Expression of CRC stem cell markers and L1 in CRC cells.
Characterization of DNA-PK and PARP-1 levels and activities in the cell lines studied. Characterization of DNA-PK and PARP-1 levels and activities in the.
SAHA blocks IR-induced increase of RAD51 protein in MM cells.
Fig. 4 Dcr-2 binds to the 3′UTR of Toll mRNA.
Effect of LPS on ERK1 and ERK2 activation in human monocytes.
Presentation transcript:

A new method to address cross-reactivity of antibodies Zestern analysis A new method to address cross-reactivity of antibodies

Cross-reactivity of antibodies One antibody, one protein (antigen) is the basis of immunoblot analysis One antibody, two or more proteins (antigens) is the reality. Majority of antibodies for research purpose actually recognize more than one protein (Cross-reactivity)*. *531 out of 22,000 antibodies are found without cross-reactivity Schwenk et al. Mol. & Cell Proteomics 9:2497-2507,2010. cross-reactivity of antibodies reflected in Western blot analysis Antibody w/o Cross-reactivity Antibody w/ Cross-reactivity

Traditional methods to address cross-reactivity of antibodies Western blot analysis: Gel electrophoresis Dot blot analysis ELISA Protein microarrays The number of samples can be analyzed are limited due to gel electrophoresis step Extensive screening to identify antibodies without cross-reactivity The number of suitable antibodies are limited. Insufficient coverage high developmental cost

Introducing Zestern analysis

Basic of Zestern analysis Excessive antigen Excessive partial antigen Synthesized epitope peptide Synthesized epitope repeat Peptide Antibody Antigen Nitrocellulose membrane Epitope

How Zestern analysis achieves specificity

Advantages over traditional immunoblot analysis Eliminates gel electrophoresis step in Western Blot analysis. Increases number of antibodies available for Dot blot, ELISA and protein microarray analysis. Increases the coverage of proteins for Dot blot, ELISA and protein microarray analyses. Serves as foundation for high throughput protein analysis. Provides basis for automation of immunoblot analysis.

Further Info. Zestern Biotechnique LLC owns US patent for Zestern analysis. PCT application has been filed by Zestern Biotechnique LLC. For further inquiry, contact: Dr. Jiandi Zhang Zesternbiotech@gmail.com (001)-703-425-6666.