80 ĐỀ THI THỬ TUYỂN SINH TIẾNG ANH VÀO 10 SỞ GD – ĐT THÀNH PHỐ HỒ CHÍ MINH NĂ...
western blot analysis for protein
1. Presented by….
RAKESH S.PARMAR
ROLL NO. :CB-0613
BATCH NO.:2013-2014
M.Sc CANCER BIOLOGY
05/09/2013
2. Blotting
Blots are techniques for transferring DNA , RNA and
proteins onto a carrier so that they can be
separated, and it often follows the use of a gel-electrophoresis
3. Blotting
techniques
Southern Blot
It is used to detect
DNA.
Northern Blot
It is used to detect
RNA.
Western Blot
It is used to detect
PROTEIN.
4. Western blotting is an immunoblotting(protein
detection) technique combining the separation
power of SDS PAGE to assess the presence, amount
and molecular- weight of proteins in cellular or
tissue extracts by using antibodies.
5. The method is characterised by transfering the protein,
which was run on a gel by electrophoresis, onto a
nitrocellulose membrane.
This approach makes the protein stable on the
membrane so that several methods including methods of
detection and quantity the protein content can be
employed.
8. Slab gel design
Comb, well
former (removed)
Sample well
Stacking gel (pH 6.8)
Separating gel (pH8.8)
Back plate
Spacer
Front plate
9. Setting up a vertical gel
Anode
Upper buffer
Compartments
Gel cassette
Lower buffer
Compartments
Cathode
10. The SDS-PAGE is sodium dodesylsulphate
polyacrylamide gel electrophoresis.
SDS-PAGE is widely used in biochemistry, forensics,
genetics and molecular biology to separate the proteins
according to their electrophoretic mobility.
Discontinuous polyacrylamide gel is commonly used as
supporting medium in separating proteins by
electrophoresis and sds (lauryl sulphate) to denature the
proteins.
11. Principle of SDS-PAGE
Proteins move in
the electric field.
Their relative
speed depends on
the charge, size,
and shape of the
protein
12. SDS is a strong anionic detergent,
means when dissolved its molecules
have net negative charge within a
wide pH range
13.
14. Polyacrylamide gels are composed of
chains of polymerised acrylamide that
are cross linked by agent like
N,N’-methylene-bis-acrylamide
15. Acrylamide and bisacrylamide used to cast the gel.
Cross links formed by bisacrylamide provides rigidity and
tensile strength to gel and forms pores through which
sds-polypeptide complex passes.
As bisacrylamide increase,the pore size decreases and
pore size increase with decrease of bisacrylamide.
16. Link
polymerization Cross Link
The polymerization reaction is catalyzed by TEMED
(Tetramethyl Ethylenediamine ) and
APS (ammonium persulfate) generates free radicals which
are necessary for the polymerization reaction
17. PAGE
Small molecules move faster than big molecules through the
polyacrylamide gel.
Bigger molecules stays near the well.
19. Uses of SDS-PAGE
Two main purposes for examining protein
preparations using SDS-PAGE
To determine the molecular weight of a protein and
To observe qualitative and quantitative differences among
the proteins from different cell preparations
Other uses include
- Identify protein
- Identify existence of disulfide bonds
- Determine sample purity
- Quantify amounts of protein
21. According to the system, preparation requires casting two
different layers of acrylamide between glass plates.
The lower layer (separating or resolving gel) is responsible
for actually separating polypeptides by size.
The upper layer (stacking gel) includes the sample wells.
It is designed to sweep up proteins in a sample between
two moving boundaries so that they are compressed
(stacked) into μm thin layers when they reach the
separating gel.
22. The samples & the stacking gel contains Tris-cl (pH6.8)
Upper & lower buffer reserviors contain Tris-glycine (pH 8.3)
Resolving gel contains Tris-cl (pH8.8)
The leading edge of the moving boundary formed by chloride
ions in the sample and stacking gel and the trailing edge is
composed of glycine molecules.
Between leading and trailing edge of moving boundary there is a
zone (gap) of lower conductivity and steeper voltage gradients
which sweeps the polypeptides from the sample and deposits
them on the surface of the resolving gel.
24. Stacking gel
To obtain optimal resolution of proteins, a stacking gel is
poured over the top of the resolving gel.
The stacking gel
-lower concentration of acrylamide (larger pore size),
- lower pH
- different ionic content
This allows the proteins in a lane to be concentrated into a
tight band before entering the running or resolving gel
produces a gel with tighter or better separated protein bands
26. Pouring of Resolving gel
Add 1/100 of fresh 10% ammonium persulfate solution. Swirl gently
to mix.
Add 1/1000 of TEMED. Swirl gently to mix.
Pour the solution into the gel cassette. Fill the cassette to a level
which will allow the comb to be inserted with 5 mm between the
bottom of the wells and the top of the resolving gel.
Overlay the gel with 1-2 mm of isobutanol to exclude oxygen and
ensure a flat interface between the resolving and stacking gels.
Allow the gel to polymerize for 30 min.
27. Pour the stacking gel solution directly onto the surface of
the polymerised resolving gel.
Immediately insert a clean Teflon comb into the stacking
gel solution ,being carefully to avoid trapping air bubbles.
Add more stacking gel solution to fill the spaces of the
comb completely.
Place the gel in a vertical position at room temperature.
30. SDS and β-mercaptoethanol denature the protein.
SDS binds with denatured proteins and covers with
negative charge
β-mercaptoethanol is a strong reducing agent. It
eliminates disulfide bonds in proteins by reducing them
(adding hydrogen atoms) and breaks apart tertiary &
quanternary structure further denaturing proteins
Dithiothreitol (DTT) is an antioxidant that breaks
disulphide bonds
31. Tris buffer maintains the pH
Glycerol or sucrose for sample density which makes settle
down the sample through electrophoresis buffer to bottom
when injected to loading well.
Ionisable tracking dye –bromophenol blue for monitoring
electrophoretic run.
32. While the stacking gel is polymerizing, prepare
the samples in the appropriate volume of 1%
SDS gel-loading buffer and heat them to 100°c
for 3 minutes to denature the proteins.
[Note: make sure to run molecular weight marker
to know the molecular weights of unknown
bands]
34. Sample loading and
Gel run
After loading samples, add electrode-buffer
to upper reservoir and lower
reservoir.
Electrode buffer provides the ions to
conduct the current through the gel.
SDS makes proteins negatively charged
that attaches the proteins to the anode.
Therefore in electrophoresis, the current
must run from cathode (negatively
charged, black) to the anode (positive
charged, red).
36. Assemble sandwitch after the gel has run
Remove gel from the glass plate holders
Place the gel in towbins buffer.
Prepare stack of whatman filter paper on blotting unit
On filter paper place nitrocellulose membrane and on top of it place
the gel.
Two type of bloting technique-submerged
in transfer buffer (Wet Blotting)
Soaked in transfer buffer (Semi dry method)
Transfer buffer contains Tris, glycine and methanol but no ions
because ions will coat the electrodes and destroy the transfer
apparatus.
Gel:nitrocellulose must have no air bubbles between them (proteins
will escape into the bubble not into the nitrocellulose.)
37.
38.
39. Second step
Transfer step
The transfer of the proteins onto the nitrocellulose
membrane.
The proteins separated on the SDS-PAGE gel are
trasferred to the membrane by using electrophoresis.
The localization of the proteins do not change.
40. Transfer apparatus
Two major types
Semi-dry apparatus
No transfer buffer chamber
Only has plates (runs for hours)
Submerge transfer apparatus
Contains a chamber filled with transfer buffer
Electrodes
-Plate electrodes (runs for hours)
-Wire electrodes (runs overnight)
41. Transfer the protein from the gel to the
membrane
Transfer of the proteins fractionated by
SDS-PAGE to a solid support membrane
(Western blotting) can be accomplished
by electroblotting
42. Running a transfer
Similar to running a gel
Negatively charged proteins run towards the anode end of the
transfer apparatus
Always have nitrocellulose on the anode side to capture proteins.
We can also use another membrane like PVDF
membrane
Why use it?
-Stronger than nitrocellulose and able to strip phosphate groups off proteins.
-Must soak in methanol first since it doesn’t become wet in water
43. Transfer
In this procedure, a sandwich of gel
and solid support membrane
(Nitrocellulose or PVDF) is
compressed in a cassette and
immersed in buffer between two
parallel electrodes.
A current is passed at right angles to
the gel, which causes the separated
proteins to electrophorese out of
the gel and onto the solid support
membrane
44.
45. How to confirm that proteins are transferred on
nitrocellulose membrane ??
Not remaining on gel or transferred more to filter paper (below membrane)
Staining gels by CBB
No bands observed
Staining
nitrocellulose
membrane by
Ponceau S dye
Orange-pink colour
bands observed
Staining filter paper
by Ponceau S dye
No bands observed
Filter paper
Gel
Membrane
Filter paper
Check by
staining
46. After transfer
Membrane is washed in a Tris buffer saline
Tween 20 solution (TBST).
Block membrane in non-fat dried milk
solution. Usually 5% w/v. Prevents
unwanted binding of antibodies to
membrane for 1 hr
Washing with TBST to remove blocking
solution
47. Third step
Primary antibody incubation step
The primary antibodies which specifically recognize the
proteins of interest are used.
48. Primary Antibodies
After blocking membrane, add primary antibodies in
concentrations recommended by manufacture or in
dilution series if unknown onto the membrane.
Incubate overnight at 4oC or 2 hours at room temperature
(only if it’s a good antibody).
49. Antibodies
Proteins that bind to specific epitopes on specific
proteins.
Two main types used in western blotting
Monoclonal
Polyclonal
Monoclonal are generally produced from mice and
polyclonal are produced from rabbit or goat but other
animals also produced polyclonals.
50. Antibodies for western blotting
Monoclonals are to one epitope while polyclonals
are to many epitopes on the protein of interest. If
epitope changes upon denaturing of the protein it
will not recognize the protein
51.
52. Fourth step
Remove primary antibody by washing with TBST
Secondary antibody incubation step.
Use of secondary antibody which recognizes the
primary antibody used in the third step.
53. Secondary Antibodies
Wash at least three times with TBST for 5-10 minutes each
Added antibodies against the animal that the primary
antibodies were made in.
These antibodies are also conjugated with enzymes such as
horseradish peroxidase
The secondary antibodies are added to the membrane and
incubated at room temperature for one hour.
54.
55. Fifth step
Visualization step
Making the antigen-antibody complex visible (staining)
Autoradiography (radioactive P / chemiluminescence)
Avidin-biotin complex
Fluoresence method.
56.
57. Detection of protein of interest
After secondary antibody incubation wash with TBST as
before.
Add enhance chemiluminance reagents (commercially
available) that have substrates for HRP that gives a product
that gives off light energy. Capture the chemiluminescence
liberated on an X-ray film
After developing the x-ray film bands will appear.
58. or for detection – colored product
The location of the antibody is
revealed by incubating it with a
colorless substrate that the
attached enzyme converts to a
colored product that can be seen
and photographed
60. Advantages of western blotting
Western blot analysis can detect one protein in a mixture
of any number of proteins while giving you information
about the size of the protein.
This method is, however, dependent on the use of a high-quality
antibody directed against a desired protein.
This antibody is used as a probe to detect the protein of
interest.
Detects proteins and estimates their molecular weight.
Detects changes in phosphorylation and lipid-modifications.
Used to detect changes in protein expression.
61. Application
1.The confirmatory HIV test
2.Western blot is also used as the definitive test for
Bovine spongiform encephalopathy (BSE(
