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Patobiochemistry, practice 1: Immunochemical detection of p53 protein on
nitrocellulose membrane (dot blot)
Aim
Immunochemical detection of oncogenic form of p53 protein
Introduction
p53 protein is encoded by TP53 gene and plays crucial role especially as a tumor suppressor. p53
regulates expression of many target genes responsible for cell growth and apoptosis thanks to its role
of transcription factor. Protein also participates in DNA reparation process. More than 50% of tumors
is associated with mutation in TP53 gene.
Dot blot is simplified version of western blot serving for detection and identification of proteins. In
difference with western blot, there is no electrophoretic separation taking place, thus it´s impossible
to determine size of target protein. Anyway, the presence of given protein (p53) can be confirmed.
Drop of the sample is loaded dirrectly onto the nitrocellulose membrane where subsequently protein
of interest can be immunochemically detected.
Principle of the method
Using of antigen-antibody reaction is principle of immunochemical detection. Antigens are
compounds recognized by organism as a foreign ones. Their presence stimulates antibody
synthesis. Every antigen consists of antigenic determinants (epitops) containing 5-8 aminoacids.
The ability of antibodies to distinguish even a small difference in epitops is basic principle of
immunodetection methods.
Antibodies fall into immunoglobulins and are produced as a part of immunity response. Several classes
can be distinguished - IgG, IgM, IgA, IgE a IgD. Antibodies are defined by an affinity – strength of
interaction between the antibody and the antigen binding determinant, an avidity – overall strength
of the antigen-antibody complex (majority of antigens is multivalent i.e. there is several binding
determinants for different antibodies) and a specifity – antibodies show only a low interference with
compounds which the antibody isn´t intend for. Antibody can be divided to polyclonal and monoclonal
ones.
Polyclonal antibodies are produced by immunisation of animals i.e. a particular antigen is applied to a
mammal. During immunisation time (2-6 months) antibodies against different antigenic determinants
of antigen are produced in blood serum. Generally, polyclonal antibody can interact with several
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antigenic determinants. On the plus side of polyclonal antibodies is higher sensitivity and avidity.
On the other hand there is an individual immune response thus irreproducibility.
Monoclonal antibodies aren´t produced by an organism but by cell culture. A mouse is immunised
by antigen then lymphocytes producing antibodies are taken out of spleen. By its hybridisation
with myeloma cells both cell types combine. By cloning only the cells producing antibodies against
particular antigenic determinant of antigen can be picked. This way produced antibody contains
only one type of binding side thus only one antigenic determinant can be recognized. Monoclonal
antibodies are characterized by higher purity, affinity, specifity and reproducibility.
Nitrocellulose membrane surface binds proteins thus blocking of membrane is necessary after
loading a sample. Membrane is immersed to some cheap protein solution e.g. BSA (bovine serum
albumine) or low-fat powdered milk in PBS with detergent addition to reduce background noise.
Proteins BSA or milk casein bind all the empty sites where proteins of our sample didn´t bind. After
antibody addition nonspecific binding to membrane surface is excluded. Antibody needs to seek
specifically its epitop placed at antigens of the sample.
Using specific primary antibody against particular protein and secondary antibody conjugated with
reporter enzyme is crucial for target protein detection. In this case, DO1 is primary antibody
recognizing specifically N-terminus of wild type protein p53 as well as its mutant form. It is about
mouse monoclonal antibody of IgG class. Secondary antibody is produced by immunisation using
particular primary antibody. Immunisation takes place in another type of host organism than it was in
case of primary antibody. In this case, DO1 primary antibody was produced in mouse thus secondary
polyclonal antibody (anti-mouse IgG) was produced in goat who was injected with primary antibody.
Overall, secondary antibody binds primary antibody and is conjugated with reporter enzyme allowing
detection, in this case with alkaline phosphatase.
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Pic. 1: Immunochemical detection of protein principle
5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitro blue tetrazolium (NBT) are commonly used for
colorimetric detection of alkaline phosphatase tagged molecules. If these compounds are incubated
with alkaline phosphatase insoluble diformazan NBT producing purple colouring is formed.
Pic. 2: BCIP/NBT reaction
Working procedure
1. Every pair has A and B sample. One of them contains oncogenic form of p53 protein while the
second one serves as a check lacking any protein. Dilute both samples 10x (23 µl of sample +
207 µl of 1x PBS).
2. Take a piece of nitrocellulose membrane, describe it with a pencil, mark sample A and B at
concentrations 1x (undiluted protein) and 10x. Vortex diluted samples. According to caption
onto the membrane load 2l of sample A and B at dilution mentioned above (see pic. 3).
Overall, there will be 4 drops onto the membrane. Touch the membrane as little as possible.
Load sample at dilution 10x first then use the same tip for loading sample diluted 1x.
1x 10x
Pic. 3: Drops of p53 at different concentration detected onto a membrane after colouring by
BCIP/NBT solution.
3. Use tweezers to place the membrane into a 15 ml falcon flask marked „milk“ and pour
approximately 5 ml of 5% PBS milk. Incubate for 15 min at shaking machine.
4. Add 1 µl of primary antibody DO1 to a milk and incubate at least for 30 min at shaking machine.
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5. Pour the milk containing antibody into a prepared 15 ml falcon flask marked „milk + DO1“ and
pour approximately 5 ml of washing buffer 1x PBST onto the membrane, leave wash for 5 min
at shaking machine. Pour out the buffer and repeat washing step twice more.
6. Pour approximately 5 ml of milk onto the membrane and add 1 µl of secondary antibody antimouse
IgG. Incubate for 30 min at shaking machine.
7. Milk containing secondary antibody pour to 15 ml falcon flask marked „milk + anti-mouse“ and
repeat washing step, wash membrane 2x after 5 min with 1x PBST and 1x with substrate
buffer.
8. Detection will be performed for three pairs together. Place 3 membranes into a Petri
dish/falcon flask. Prepare developing solution by adding 33 µl of BCIP and 330 µl of NBT to 10
ml of substrate buffer. Incubate membrane in this the solution at shaking machine as long as
purple colouring observed (about 3 min).
9. Stop the reaction by washing with distilled water.
Used chemicals
1x PBST – 137 mM NaCl, 12 mM phosphate, 2,7 mM KCl, 0,05% Tween, pH 7.4
5% PBS milk – 5% powdered milk in 1% PBS
BCIP – 50 mg/ml water solution
NBT – 10mg/ml water solution
Substrate buffer – 0,1 M Tris, 100 mM NaCl, 5 mM MgCl2 , pH 9,5
Evaluation
Determine which sample contains oncogenic form of p53 protein.
Report
Name:
Date:
Method principle:
Conclusion:
Questions:
1. What do you know about p53 protein?
2. What is dot blot?
3. Explain immunochemical detection of protein principle.
4. What is milk/BSA used for during immunochemical detection of protein?
5. Which enzyme catalyze purple coloured diformazan NBT formation?