Antibody methods Flashcards
What is the importance of proteins?
Function as:
Transcription factors
Signals
DNA and RNA replication
Catalysts
Receptors
Intracellular signalling
Structural
How can you determine where the protein is in the cell?
Immunohistochemistry or immunofluorescence.
How do you determine how much protein is in the cell?
SDS-PAGE and then western blot.
How do you determine how much protein is in the fluid?
ELISA
How do you determine if cells secrete the protein?
ELISPOT
How do you identify the protein?
Weight - separate the proteins on a gel.
But many proteins weigh the same, so identify using a specific antibody.
In tissue, through ELISA, ELISPOT or through a gel and then western blot.
What is the structure of antibodies?
Variable regions - Y shape, sticks to the antigen.
Constant region - place where things can be attached to.
What are antibody sandwiches?
ELISPOT, ELISA and western blotting use antibodies to bind to a protein and then amplify a signal.
Why are antibody’s and proteins amplified?
Epitopes are very small and may only get one per protein.
Antibodies are also very small.
A big signal is needed to see anything.
What is the primary antibody?
Binds to the epitope first.
The antibody that binds to the primary antibody is the secondary antibody.
The secondary antibody must be specific to the species that the primary antibody came from.
What is the normal immune response?
If a foreign substance or antigen is injected into a vertebrate, the B cells will turn into plasma cells and produce antibodies against the antigen.
Each B cell will produce structurally different antibodies (polyclonal) that bind to different parts of the antigen, so are very versatile.
These antibodies can be isolated from the blood and used in research.
How can polyclonal antibodies be removed?
Immunise a rabbit with a peptide from the protein you want an antibody from.
Then either:
Bleed the rabbit, let it clot, centrifuge, and the serum will contain the structurally different antibodies.
Or remove B cells from the spleen, which become plasma cells and produce antibodies into blood.
What are the limitations of polyclonal antibodies?
They can only be used once and there is a limited supply from the animal.
Ideally want an infinite supply with limited suffering.
There is more chance of background non-specific detection.
How are monoclonal antibodies produced?
Immunise mouse with the peptide.
Isolate the B cells from the spleen. Then fuse with myeloma cells (antibody producing cells)grown in culture to form hybridomas.
Hybridomas are screened, and those producing antibodies are cloned and expanded, then purified.
What are the benefits of monoclonal antibodies?
The hybridoma can keep growing forever.
There is only one type of antibody which binds to one bit of the protein, so is very specific.
It can be used for everything.
There is less chance of background non-specific detection.
What are the limitations of monoclonal antibodies?
Expensive and requires hybridoma technology.
If the epitope of the antigen is lost e.g. in Western Blotting then the antibody wont work.
What is ELISA?
Enzyme linked immunosorbent assay
Looks at what’s in fluids - blood, urine, saliva, cell culture media.
How does ELISA show how much protein is in a fluid?
The darker the colour change, the more protein.
The sample is diluted down the plate to have accurate data.
What is the process of preparing ELISA?
Coat the wells with a primary antibody specific to the protein.
Block the wells with non-specific protein - BSA, cheap.
Add fluid of interest so the protein can bind to the antibody.
What is the process of testing the protein in ELISA?
Add your detecting (secondary) antibody (has enzyme attached) to bind to the protein at a different epitope (position).
Use a chemical substrate for the enzyme - the colour will change depending on how much secondary antibody binds.
Measure the colour change using a spectrophotometer.
Why is BSA added?
The wells are sticky to bind to your primary antibody so need to stick something to it to fill any gaps.
What are the requirements for ELISA?
Need repeats.
Need a dilution series to avoid over and under saturation.
Have a negative control - wells that have defined amount of protein in them.
In between steps the plate needs washing with PBS and tween - a detergent.
How is the amount of protein in ELISA calculated?
Use your protein standard row to calculate the amount of the protein of interest.
Or use your control group for comparison.
What does an ELISPOT plate look like?
It has a white background.
The ELISA plate was clear because light was shined through to measure absorbance.
This has a membrane to put antibodies on, so is white to see them better.
What is ELISPOT?
Used to determine if the cells are producing something e.g. growth factors, antibodies, hormones and cytokines.
What is the process of ELISPOT?
Coat your plate with an antibody then block it.
Add your cells and incubate them to produce proteins.
After incubation, wash your plate to remove the cells.
Then add the secondary antibody which is bound to an enzyme.
Then add the substrate for the enzyme.
It will form precipitates - each cell producing the protein will form a dot.
What happens when there is no colour change or spots in ELISA and ELISPOT?
The wrong antibody may have been used.
The reagents may have gone off.
Too much or too little signal - use more or fewer cells, or more or less fluid - by titrating the antibody reagents or fluid.
What are the methods for determining how much protein is in the cell?
SDS-PAGE and then western blotting.
PAGE = polyacrylamide gel electrophoresis.
What is the process of determining how much protein is in the cell?
Get the proteins out of the cell or supernatant
Measure how much protein there is.
SDS-PAGE - linearise the protein then separate out by weight.
Find the protein using Western Blot.
How are proteins isolated?
Detergent reacts with membrane, dissolves it, then intracellular components are released.
Common detergents are Tween, Triton, NP-40.
How do you then determine how much protein there is isolated?
Need to use the same amount of protein from each sample.
Use Coomassie-blue to react with the proteins, the more brown the more protein there is.
Use a spectrophotometer, proteins are measured at 280nm.
How is a protein standard curve used to determine how much protein there is isolated?
The known concentration of protein is diluted so there is a precise amount in solution.
Add Coomassie-blue, then record the absorbance at the wavelength to make an absorbance concentration curve.
Then can use a precise equation to known exactly how much protein is in the sample so the reagent concentrations are correct for western blotting.
What is protein gel electrophoresis?
Polyacrylamide gel - a mixture of acrylamide and bis-acrylamide.
Altering the ratio between the reagents changes the pore size - high molecular weight proteins are separated at low %gel.
The total acrylamide concentration in the gel affects the migration of proteins through the matrix.
What is SDS-PAGE?
Sodium Dodecyl Sulphate polyacrylamide gel electrophoresis.
It is a denaturing gel so the proteins are linearised and there is only one molecule.
What is the preparation of the sample for SDS-PAGE?
Measure equal amounts of proteins, in gel.
Add denaturing buffer that has SDS, binds to proteins and gives it a negative charge, so it is separated only be molecular weight.
The amount of charge depends on how much protein there is.
Add 2B-mercaptoethanol to break disulphide bonds.
Add glycerol so samples are more dense and sink into the well.
Add bromophenol blue so can track the sample, and indicate pH - yellow/brown = acidic so needs neutralising.
What happens to the sample for SDS-PAGE for loading?
Heat it to 95 degrees for 10 minutes, then place on ice.
This breaks weak hydrophobic bonds.
The sample is now charged, linear proteins.
What is the gel in SDS-PAGE?
It is a vertical thin gel with two types.
Stacker gel and Resolving gel.
There is also a running buffer of Tris: Glycine.
Glycine runs slower than the slowest protein.
What is the stacker gel?
It has Tris/HCl at pH 6.8.
It is large pore acrylamide.
The samples are loaded onto, and the proteins are concentrated.
What is the resolving gel?
It has Tris/HCl at pH 8.8.
It is small pore acrylamide.
This separates out the proteins by size.
What does the SDS-PAGE electrophoresis buffer have?
Tris-HCl to maintain pH.
SDS to maintain denaturation.
Glycine.
How are the proteins visualised after SDS-PAGE?
Coomassie-blue stain shows all the proteins, it is a good control.
Silver stain can detect low amounts of proteins, but the gel cant be used pass this point.
A ladder is required to know how big the proteins are.
Doesn’t tell you about the particular protein so then western blot.
What is Western blotting?
After the PAGE:
Verifies that the protein has been expressed.
Determines the relative amount of a protein.
Analyses protein-protein interactions.
What are the types of Western Blots?
Denaturing - SDS-PAGE.
Non-denaturing - native PAGE, so the proteins stay in their complexes.
What is the problem with SDS-PAGE?
The gels are very flimsy so antibodies can’t be put straight onto the gel.
Instead proteins are transferred onto a more robust membrane.
How is the Western Blot constructed?
The gel is put on a membrane, with filter paper on either side.
Filter paper is then sandwiched between the electrodes.
The reagents need to be wet so that the proteins can transfer between gels.
The proteins are negative, so go the anode, so the membrane needs to be on the correct side of the gel.
See picture.
How are the proteins transferred in Western Blotting?
The electric current pulls proteins from the gel into the nitrocellulose of PVDF membrane.
Transfer buffer is used, containing Tris, Glycine and methanol.
Transfer can then be checked using a non-permanent stain - Ponceau S.
What are the steps involved in Western Blotting?
Blocking.
Incubation with primary antibody, then washing membrane.
Incubation with secondary antibody, then washing membrane.
Incubation with enzyme substrate.
Exposure to photographic film/ detector.
What is blocking in Western Blotting?
Block the membrane with non-specific proteins.
The proteins bind to unbound regions of the membrane.
It can then be incubated with the primary antibody, which will only bind to the specific protein.
This avoids false positives.
What are the problems of blocking?
If the blocking is too high there will be a low signal.
If the blocking is too low there will be a high background.
What materials can be used for blocking?
Bovine serum albumin
Casein
Gelatin
Ovalbumin
Fat-free milk, which is often used because it is the cheapest, and can be used for lots of proteins so is efficient.
What is the primary antibody incubation?
After blocking, the primary antibody is diluted in blocker.
Dilution is based on its titre - if titre against target protein is high (good binding) then increase dilution before applying to the membrane.
Incubate for 1-2 hours at room temperature.
What is washing the membrane in Western blotting?
Washed with PBST, more than once, to get rid of primary antibody.
The time and number of washes is antibody dependant.
Usually 3 x 15 minutes at room temperature.
What is PBST?
Phosphate buffered saline + Tween 20
What is incubation with the secondary antibody?
The secondary antibody is diluted in blocker.
Incubation varies, normally 5 minutes to 1 hour.
The antibody must bind to the primary e.g. anti-rabbit to bind to a rabbit primary.
Secondary antibody is needed to amplify the signal, so the antibody can be visualised.
What are the drawbacks of Western Blotting?
Many steps where errors can occur.
Large amount of sample needed
Semi quantitative through comparing bands.
Time consuming protocol
Cannot give localisation data, need to use immunofluorescence on slides.
- The protocol calls for a solution that is 1.5 % (w/v) NaCl, 2mM Tris (pH 8.0), 7% (w/v) powdered milk and 0.02% (v/v) Tween 20. Work out how to make up the solution of 1L.
1.5g of NaCl per 100ml.
For 1000ml need 15g.
2mM Tris, molar mass 121.14g/mol.
Mass = molarity x volume x molar mass.
0.002M x 1L x 121.13g/mol = 0.242g.
7g powdered milk per 100ml.
1000ml = 70g milk.
0.02ml of Tween per 100ml.
1000ml = 0.2ml Tween.
Dissolve 15g of NaCl, 0.242g Tris, 70g powdered milk in 800ml distilled water, add 0.2ml Tween and mix.
Then bring total volume to 1000ml with distilled water.
What can go wrong in Western blotting?
Incomplete transfer of proteins from the gel to the membrane.
Uneven blocking, causing nonspecific binding - background noise.
Insufficient washing, leaving residual antibodies.
Protein loaded differently.
Membrane may have defects or folds.
