Blotting, gels and samples
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Blotting, gels and samples:
Western blotting relies on crude, purified and semi-purified extracts from cell proteins containing the protein of interest to be detected by the antibody. In order to accomplish this, several key steps have to be taken so that the prepared protein is stable and does not impact the results in any way.
The three steps of preparation are:
1. Producing a sample via lysis or homogenization in order to solubilize and later release the cell proteins.
2. Separating protein mixtures by gel electrophoresis.
3. Moving the isolated proteins on a blotting membrane, which is easier to manipulate.
The three steps of preparation are:
1. Producing a sample via lysis or homogenization in order to solubilize and later release the cell proteins.
2. Separating protein mixtures by gel electrophoresis.
3. Moving the isolated proteins on a blotting membrane, which is easier to manipulate.
Sample preparation
Crude cell lysates are the most common pick for starting material in Western blotting. They are derived from immortalised cell lines expressing the protein of interest or alternatively from transfected cells, which have the protein expression vector.
Usually, the cells have to be washed and lysed in order to release the protein of interest. Minimizing proteolysis, denaturation and dephosphorylation by conducting these steps on ice helps with keeping the reactants in good condition.
Choosing the right lysis buffer is sometimes a hard task, with only the type of isolated protein being a guide. These buffers range from very gentle ones, completely lacking detergent, to much harsher ones, containing numerous detergents and denaturing properties. Furthermore, during lysis the interaction between the antibody and the antigen can change, which should be considered then performing this step in order to ensure reliable results.
Usually, the cells have to be washed and lysed in order to release the protein of interest. Minimizing proteolysis, denaturation and dephosphorylation by conducting these steps on ice helps with keeping the reactants in good condition.
Choosing the right lysis buffer is sometimes a hard task, with only the type of isolated protein being a guide. These buffers range from very gentle ones, completely lacking detergent, to much harsher ones, containing numerous detergents and denaturing properties. Furthermore, during lysis the interaction between the antibody and the antigen can change, which should be considered then performing this step in order to ensure reliable results.
Purified and semi-purified cell extracts
A basic source of material for Western blotting is protein samples, both purified and semi-purified, which are derived from the protein purification process. Their main advantage is that they require little to no manipulation before being added to the gel electrophoresis loading buffer.
When these types of extracts, a much smaller amount of total protein can be added to the gel.
When these types of extracts, a much smaller amount of total protein can be added to the gel.
How to determine protein concentration?
In order to have the samples in the suitable detection range for the specific assay, so they can be measured on the same basis, the concentration of total protein must be noted. Nowadays, several kits and reagents exist, making this process much more reliable.
First, measuring the absorbance of the lysate at either 205 nm or 280 nm can be enough. Another option is to use a metal ion reduction on the peptide bond assays or to use dye binding, in the case of the Bradford assay.
First, measuring the absorbance of the lysate at either 205 nm or 280 nm can be enough. Another option is to use a metal ion reduction on the peptide bond assays or to use dye binding, in the case of the Bradford assay.
Loading buffer
When protein concentration has been determined, samples are diluted in gel loading buffer. This buffer ensures that the samples sink easily into the wells of the gel, and a tracking dye, which migrates through the gel first to indicate how far the separation has progressed is also present. Usually, SDS and a reducing agent are also present in the gel loading, to ensure full denaturation of the protein and remove all higher order structure. Samples are then heated in gel loading for either 5 minutes at 100 °C, or 10 minutes at 70 °C to help with denaturation.
Standards and controls
Having a positive and a negative control on the gel along with the samples that are being evaluated is quite useful. For a positive control, using a known source of target protein, such as purified protein or a control lysate is advisable. The positive control confirms the identity of the target since it produces a reference band on the blot showing the expected migration of the target protein and confirming the activity of the antibodies. Also, including negative sample control like a known null cell line, to confirm that the signal is specific to the protein of interest is a good measure. Lastly, a molecular weight standard is needed since the main role of Western blotting is to provide information on the size of the protein. Molecular weight standards come in many formats, ranging from unstained, pre-stained, multi-coloured, or directly labelled for Western detection. Monitoring progress while the gel is running, checking transfer efficiency, orienting the immunoblot can all be fail proofed using this standard.
Gel electrophoresis
Following the sample preparation, they are separated according to size using SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). In the previous step of denaturing the samples in a SDS detergent buffer, the protein has been negatively charged and can now move through the gel to the positive electrode.
After the gel sets, it can be placed in the apparatus and small volumes of gel with dissolved protein are then added to every well. Following that, the power supply is connected to the gel and inside a buffer tank the gel is left to separate the proteins inside.
After the gel sets, it can be placed in the apparatus and small volumes of gel with dissolved protein are then added to every well. Following that, the power supply is connected to the gel and inside a buffer tank the gel is left to separate the proteins inside.
Blotting
There are two types of solid support onto which the separated proteins are transferred, nitrocellulose or polyvinylidene fluoride (PVDF) membrane, both bind proteins with high affinity.
Considering the size of the target protein is important. Smaller proteins will transfer out of the gel faster. After the blotting step is completed, the apparatus is carefully disassembled, and the transfer is examined. The easiest method of confirming if the transfer is successful involves the appearance of pre-stained markers on the blot as compared to the gel. But this only provides results for a single lane.
A better method of confirming the successful transfer is using a reversible stain which identifies if there are protein bands on the membrane.
Considering the size of the target protein is important. Smaller proteins will transfer out of the gel faster. After the blotting step is completed, the apparatus is carefully disassembled, and the transfer is examined. The easiest method of confirming if the transfer is successful involves the appearance of pre-stained markers on the blot as compared to the gel. But this only provides results for a single lane.
A better method of confirming the successful transfer is using a reversible stain which identifies if there are protein bands on the membrane.