Transcript Western blot, fehérjék elektroforézise, fehérjechip

Western blot,
Protein electrophoresis, ELISA,
Protein chip
From DNA to protein
Why it is easy to separate DNA?
The structure of proteins
The
classes
of aminoacids
3D structure of protein
The isolation of proteins
Cell Lysis
Freeze/thaw and homogenization. Special detergent-based reagents
Affinity Purification
Centrifugation of crude cell lysate. Affinity chromatography. Magnetic particles.
Sample Preparation
Purity and concentration checkup
The methods described apply to preparation of samples for use in many common
laboratory techniques such as electrophoresis, Western blotting, ELISA, mass
spectrometry, enzyme activity assays and more.
Antigen - antibody
Antigens: objects, recognized as foreign by vertebral
organisms and can challenge the immune system to produce
specific antibodies and immune cells against antigens. The
order of immuno-stimulant strength: proteins >polysaccharides
> lipids > nucleicacids.
Antibodies: Immunoglobulins produced by the B cells of the
immune system. They recognize and react with the antibodies
specifically. The antibodies can be separated from the
gammaglobulin fraction of the blood serum.
Methods based on specific antigen and
antibody binding
Direct method
We label the antigen or the antibody
Indirect method
We detect the antigen-antibody binding with a labelled anti-immunglobulin antibody (e.g. goat
anti-human IgG) that recognize the specifically reacting primary antibody mutatjuk ki. Method
is mainly applied to detect antigen specific antibodies and for their. Increased specificity.
Double antibody: „sandwich” method
In this method we bind the antibody-molecules reacting specifically with the antigen to solid phase.
The anchored antibody specifically binds the antigen, thus the antigen isolated from
multicomponent solution. The antibody-antigen binding is the detected by another specifically
reacting labelled antibody.
The essence of Western-blot
Acrylamide gelelectrophoresis
Pretreatment of protein sample for running (denaturation)
Before the electrophoresis add detergent (SDS, sodium-dodecilsulfate)
and disulfide bridges reducingagent (mercaptoethanol) to samples
and apply heat treatment (3-5 min., 90°C).
Polyacrilamide electrophoresis
Denaturation gel with SDS
Two layers: a concentrating and a separation gel layers
Electroblot
Vertical gel
Checking the efficiency of blotting
Coomassie-brilliant-blue dye (CBB)
Acrylamide polymerization
Caution: The acrylamide and bis-acrylamide are neurotoxic!!!
The effect of SDS pretreatment
Before SDS
Charged parts
Hidrophobe parts
After SDS
Vertical gel set up
Running a protein separation gel
Staining the protein gel
STANDARD PROTOCOL
Blocking of membrane ( blot )
To saturate nonspecific protein binding sites, incubate the nitrocellulose and PVDF membranes membrane for 30-60
minutes in Blocking Buffer ( TBST containing 1% BSA or 5 % skimmed milk).
Primary Antibody Binding
1.To add primary antibody, replace the blocking solution ( which can be re-used several times ) with Blocking Buffer
containing appropriate dilution of primary antibody. Incubate the blot for 30 ~ 60 minutes with gentle agitation at
room temperature (or overnight at 2~ 8 °C).
2.To remove unbound antibody, wash the membrane three times with TBST for 5 ~ 10 minutes each.
Secondary Antibody Binding
1.Incubate blot with Blocking Buffer /Antibody Diluent containing the appropriate antibody dilution (e.g.goat antihuman IgG-HRP conjugate) for 30 minutes.
2.Wash the blot with TBST three times for 10 minutes each to remove unbound secondary antibody.
Development of signal
Add alkaline phosphatase subtrate, HRP substrate, ECL substrate
Blotting
Checking the efficiency of blotting on
membrane: Ponceau staining
The way from gel separation to detection
of the wanted protein
ECL detection
ECL: Enhanced Chemiluminescence
Alkaline phosphatase detection
Application of Western blot for Lyme
disease detection
Detection of bacterial proteins of Borrelia burgdorferi spreaded by tick
byte in a patient blood sample
Lyme disease reactive Western blot
Description of lanes:
Lane 1 - molecular weight marker
Lane 2 - positive patient sample
Lane 3 - positive patient sample
Lane 4 - monoclonal antibodies for
39 and 41kD bands
Lane 5 - monoclonal antibodies for
41kD band
Lane 6 - monoclonal antibodies for
39 and 41kD bands
Lane 7 - monoclonal antibodies for
31 and 34kD bands
Lane 8 - positive control pool
Immunoblotting (western blotting) detects
proteins that have been size-fractionated on
an electrophoresis gel
Protein chip
Ligand-chip: It is possible to detect protein pattern of a cell
by using a method based specific antigen antibody binding
(at DNS chips the hybridization is the basic technique)
More hundred antibodies that can be immobilized on a solid
surface applied with the help of a robot in great density on
activated glass carrier. The control of chips is performed
with widely used method checking the interaction ligandprotein pair, determining level of aspecific binding and the
background.
Application for research and diagnostic (eg. monitoring the
alteration in protein pattern of tumorous patient).
Detection on protein chip
Yeast protein pattern on protein chip
Automatization
ELISA
The ELISA (Enzyme Linked Immunosorbent Assay) is an immunotest
with high sensitivity, in which the antigen or the antibody is linked to a
solid (plastic) surface. The test generally performed in plastic plates with
96 wells (in 100-200 l volume). Mostly, the the antigen is pre-absorbed to
the plastic surface then different dilutions of the tested serum sample (from
a patient) are added to the wells.
Application: The application scale of ELISA is broad (e.g. Identification
of viral and bacterial infections; the identification and quantification of
hormones or cytokines in blood circulation, etc). The antibodies produced
against pathogenic microorganism can be identified in blood. An infection
state can be deducted from the increase or decrease of specific antibodies.
The test sensitivity is high, ng amount can be measured.
ELISA data from three patients
Positive
Control
Negative
Control
1.689
0.153
Patient A
Patient
B
Patient
C
Assay
Control
O.055
0.412
1.999
0.123
Partially purified, inactivated HIV antigens pre-coated onto an
ELISA plate
Patient serum which contains antibodies. If the patient is HIV+,
then this serum will contain antibodies to HIV, and those
antibodies will bind to the HIV antigens on the plate.
Anti-human immunoglobulin coupled to an enzyme. This is the
second antibody, and it binds to human antibodies.
Chromogen or substrate which changes color when cleaved by the
enzyme attached to the second antibody.
1. Which of these steps do not belong to a Western-blot?
a) Denaturation
b) Blocking
c) Hybridization
d) Antibody binding
e) Development
5. Which component is not needed for a Western-blot?
a) SDS
b) mercaptoethanol
c) probe
d) nitrocellulose filter
e) agarose gel
3. Which statement is not valid for antibodies?
a) They belong to proteins
b) They can bind only specifically
c) Can be found in mother milk, too
d) they have two antigen binding sites at least
e) Generally can be isolated without previous immunization