Master’s seminar
Biochemical and Immunological characterization of
polyclonal antibodies generated against RIBEYE, Tctex1, Cav1.4 and Cre protein in retina
15.07.2015
Praveen Kumar
Under guidance of
Prof. Dr. Frank Schmitz
Institute for Anatomy and Cell Biology
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CONTENTS
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Aim
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Introduction
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Molecular and biochemical techniques
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Application
•
Benefits
•
References
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AIM
Aim of this project is to express and purify RIBEYE, Tctex-1,
Cav1.4 and Cre-recombinase fusion protein and to generate
polyclonal antibodies against these proteins. Further, these
antibodies will be used for various Biochemical and
Immunological characterization of above mentioned
proteins in retina.
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INTRODUCTION
THE VISUAL SYSTEM
The part of the central nervous system, which interprets the information from
visible light to build a representation of the world surrounding the body.
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RETINA
There are six classes of neuron in the mammalian retina: rods (1), cones (2), horizontal cells
(3), bipolar cells (4), amacrine cells (5) and retinal ganglion cells (RGCs) (6). They have a
laminar distribution (OS/IS, outer and inner segments of rods and cones; ONL, outer
nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform
layer; GCL, ganglion cell layer; NFL, optic nerve fibre layer)
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SYNAPTIC RIBBONS
Ultrastructure of synaptic ribbons.
(A) Conventional transmission electron micrographs of the photoreceptor synapse from (Carassius carassius).
Synaptic ribbon (sr), synaptic vesicle (sv), horizontal cell (hc) and bipolar cell (bc). The synaptic vesicles are
shown by arrows; the arrowheads denote the site of endocytosis (Schmitz, 1996).
(B) A synaptic ribbon (sr) is tangentially cut, thus making the plate-like character of synaptic.
sr = synaptic ribbon; sv = synaptic vesicle; pr = presynaptic terminal; en = large, tubular compartment
possibly of endosomal origin; ad = arciform density; sri = synaptic ridge; hc, bc = horizontal/bipolar cell
dendrites. Scale bars: 100 nm (A, B).
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RIBEYE, a component of synaptic ribbons
RIBEYE is composed of a unique A
domain specific for ribbons, and a B
domain identical with
transcriptional repressor.
CtBP2,
a
Evolved in vertebrates under utilization
of a preexisting protein to build a unique
scaffold for a specialized synapse
A domain: Mediates assembly of Ribeye
into large structures.
B domain: Binds to NAD(H) with high
affinity.
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CaV1.4 calcium channels
Cellular function:- Release neurotransmitter from photoreceptors, mediate the
entry of calcium ions into excitable cells.
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TCTEX-1, a light chain of the dynein complex
The major microtubule minus end–directed motor protein of the cell; it is
involved in many essential cellular processes such as membrane trafficking and
mitosis.
Function:
(1) end-directed organelle movement,
(2) endosomal transport ,
(3) centrosomal localization of the Golgi complex, (4) mitotic spindle alignment,
(5) anaphase chromosome segregation,
(6) and nuclear distribution,
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CRE-RECOMBINASE
- Cre- recombinase, often abbreviated to Cre, is a Type1 topoisomerase form P1
bacteriophage that catalyzes site specific recombination of DNA between loxP
sites.
- Does not require any energy cofactors and Cre-mediated recombination, quickly
reaches equilibrium between substrate and reaction products.
- Capable of creating any desired modification of the mouse genome (addition of
functional genome pieces, remove exciting gene from genome, excise or invert
loxP flanking DNA segment, create intramolecular recombination between
different DNA molecules)
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MOLECULAR AND BIOCHEMICAL
TECHNIQUES
6/8/15
(A) Cloning, Expression and Purification of Crerecombinase
PCR DNA Amplification
DNA cloning into plasmid
Transformation of competent BL21 bacteria
with different techniques(heat shock,
electroporation)
Expression and purification of BL21 bacterial
fusion proteins
Protein quantification with colorimetric assay
such as Brodford assay
6/8/15
(A) Cloning, Expression and Purification of Crerecombinase
Gel images of plamid vector pMAL-C2 (6626bp) contain Cre-recombinase
DNA insert (1Kb). Showing successful cloning in 20 E.coli colonies whose chosed
for heterologous expression.
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(A) Cloning, Expression and Purification of Crerecombinase
6/8/15
(B) GENERATION AND CHARACTERIZATION OF
POLYCLONAL ANTISIRA
Generate anti rabbit polyclonal antibodies using
fusion protein
Identify specific proteins from a complex mixture
of
proteins extracted form rabbit serum by using
western blotting
IMMUNOHISTOCHEMISTRY OF RETINA TISSUE
Visualize the location of proteins on
tissue
sections
by
using
immunofluorescence
Ribeye
2D9
Ribeye+ 2D9
merge
phase
ONL
OPL
INL
APPLICATION
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Research applications such as: western blot, ELISA, flow cytometry,
immunohistochemistry, immunofluroscence etc.
Disease case studies such as: Retinitis pigmentosa, Night blindness,
Blind vision, Myopia, retinal degeneration, color vision defect etc.
Pathway studies such as: signal transmission, membrane
depolarization, cell death, T-cell activation, transport, cell activation,
muscle contraction, phototransduction, segmentation, lymphocyte
activation and differentiation etc.
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BENEFITS



Provide beneficial antibodies at affordable prices and in large amount.
Check reactivity in variety of model species such as: Human, Mouse,
Rat, Bovine, Porcine etc.
Open new areas of research in disease diagnosis and therapeutic drug
studies.
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REFERENCES
 Alpadi, K., Magupalli, V.G., Käppel, S., Köblitz, L., Schwarz, K., Seigel, G.M.,
Sung, C.H and Schmitz, F. (2008). RIBEYE recruits Munc119, a mammalian
ortholog of the C. elegans protein unc119, to synaptic ribbons of photoreceptor
ribbon synapses. J. Biol. Chem. 283, 26461-26467.
 Mohammad Hossein Maghami, MS; Amir Masoud Sodagar, PhD; Alireza Lashay,
MD Hamid Riazi-Esfahni, Mohammad Riazi-Esfahani, MD. Visual Prostheses:
The Enabling Technology to give Sight to the Blind.
 Jean Defourny , François Lallemend , Brigitte Malgrange. Structure and
development of cochlear afferent innervation in mammals, American Journal of
Physiology - Cell Physiology Published 1 October 2011 Vol. 301 no. 4, C750-C761
DOI: 10.1152/ajpcell.00516.2010
 Andrew W. Tai, Jen-Zen Chuang and Ching-Hwa Sung. Localization of Tctex-1, a
Cytoplasmic Dynein Light Chain, to the Golgi Apparatus and Evidence for
Dynein Complex Heterogeneity
 http://www.ptglab.com/Support/TechnicalSupport/LearningCenter/AntibodyPr
oductionAndPurification.aspx
 http://info.gbiosciences.com/blog/topic/western-blotting
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REFERENCES
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http://www.epubbud.com/read.php?g=UC5RFDTJ&p=1
http://www.corpshumain.ca/en/Vue_en.php
http://tigger.uic.edu/orgs/psych/pages/l2/sld022.html
http://www.ncbi.nlm.nih.gov/pubmed/11163272
http://www.science.oregonstate.edu/~barbare/research.html
http://en.wikipedia.org/wiki/Cre_recombinase
http://www.accessexcellence.org/RC/VL/GG/plasmid.php
http://www.seekbio.com/biotech/exp/Immunity/Antibody/2011/o8103656112.html
http://www.rndsystems.com/resources/images/6295.gif
http://www.ruf.rice.edu/~rur/issue1_files/Biology/Norman/Norman_fig2.jpg
http://www.uniprot.org
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