Western PDF

Western blot,
Protein electrophoresis,
ELISA
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From DNA to protein
Based on its structure the DNA is not
good antigen
The structure of proteins variable and
complex
The
classes
of amino-
acids
Examples of 3D structure of protein
Tubulin
dimer
Haemoglobin
GFP
The isolation of proteins
Cell Lysis
Freeze/thaw and homogenization. Special detergent-based reagents (It is
important to block the proteases!)
Affinity Purification
Centrifugation of crude cell lysate. Affinity chromatography. Magnetic
particles.
Sample Preparation
Purity and concentration checkup
The described methods can applied to prepare samples for many commonly
applied laboratory techniques: such as electrophoresis, Western blotting,
ELISA, mass spectrometry, enzyme activity assays, etc.
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 isolated 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 directly
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 main steps 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 gel-electrophoresis (PAGE)

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 treatment on proteins
Before SDS
Charged parts
Hidrophobe parts
After SDS treatment
Vertical gel set up
Sample
Gel
pockets
Buffer
Gel
Buffer
A protein separation gel
Staining of the protein gel
PROTEIN DETECTION ON THE FILTER
Blocking of membrane ( blocking)

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 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 anti-human 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
Transfer to membrane made of nitrocellulose or polyvinylidene difluoride (PVDF)
Checking the efficiency of blotting on

membrane: Ponceau staining
Steps from gel separation to detection of
the wanted protein
ECL detection
ECL: Enhanced Chemiluminescence
Alkaline phosphatase detection
NBT, BCIP
(colorless)
NBT (reduced),
IgG antibody AP BCIP(dephos.)
conjugates violet-blue
color
Specific antibody
(IgG) Membrane
Protein
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
41kD band
39 and 41kD bands
31 and 34kD bands
Lane 8 - positive control pool
Semiquantitative immunoblotting
(western blotting)
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.
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.
A HIV Test
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.
The test process
The evaluation of an ELISA plate
ELISA data of three
patients
Positive Negative Patient Patient Assay

Control Control Patient A B C Control
1.689 0.153 O.055 0.412 1.999 0.123
ELISA reader

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Western blot,

Antibodies: Immunoglobulins produced by the B cells of the

Double antibody: „sandwich” method

Polyacrilamide gel-electrophoresis (PAGE)

Checking the efficiency of blotting

Caution: The acrylamide and bis-acrylamide are neurotoxic!!!

After SDS treatment

Blocking of membrane ( blocking)

Primary Antibody Binding

Secondary Antibody Binding

Checking the efficiency of blotting on

Detection of bacterial proteins of Borrelia burgdorferi spreaded by tick

The application scale of ELISA is broad:

Partially purified, inactivated HIV antigens pre-coated onto an

Patient serum which contains antibodies. If the patient is

Anti-human immunoglobulin coupled to an enzyme. This is the

Chromogen or substrate which changes color when cleaved by

Positive Negative Patient Patient Assay

1.689 0.153 O.055 0.412 1.999 0.123

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