The Western Blot is an immunoassay for the detection of proteins in complex samples that is carried out following 4 sequential steps:
- SDS-PAGE (polyacrylamide gel electrophoresis) to separate proteins.
- Protein transfer to a nitrocellulose or polyvinylpyrrolidone membrane.
- Incubation of the membrane with a specific antibody against the protein of interest.
- Detection of antigen-antibody binding.
In this post we bring you a compilation of frequently asked questions about Western Blot that can help you answer questions and improve the performance of your immunoassay.
1.- How many times can I use the antibody solutions once they are prepared?
It is recommended that you always use freshly formulated antibody solutions. In the event that previously made solutions are reused, it is of utmost importance to ensure that no bacterial overgrowth has occurred, especially in cases where the solution has previously been blocked with a blocking agent.
2.- Why do intense bands appear at higher or lower molecular weights than expected?
On some occasions, even using the antibodies at the lowest recommended dilution, it binds to proteins whose bands come out well below or well above the actual molecular weight of the protein of interest. In most of these cases, these bands correspond to isoforms of the protein in question, or to the formation of dimers.
A search is recommended to see if any isoforms are described in the literature or if the protein dimerizes. The use of a different antibody can also be tested.
3.- If you use more than one primary antibody, in what order should they be incubated?
Both simultaneous incubation with all primary antibodies and successive incubation with each is possible.
4.- Is it necessary to measure the protein concentration in the sample before doing the Western Blot?
It is not an essential step, although it is recommended to adjust the amount of sample to be loaded in the gel. The determination of the total protein concentration can be carried out by the BCA method .
5.- How can I separate and transfer proteins with sizes greater than 200kDa?
You can find several tips for transferring large proteins in this post .
6.- How can I avoid background noise?
This is one of the most common western blot frequently asked questions. Background noise may be due to causes as disparate as too high an antibody concentration, nonspecific binding of the secondary antibody, cross-reactions of the antibodies with the blocking agent or insufficient washes, among others.
Here we tell you how to solve these problems in Western Blot.
7.- Is Western Blot a quantitative immunoassay?
The Western Blot is not a quantitative method, since a standard curve for the protein of interest is not usually performed in each blot.
8.- Why are the Western Blot bands different in size than expected?
Although the separation of proteins in the Western Blot is based on their size, there are other variables that can influence the migration speed through the gel, and cause the observed band to differ from what could be predicted based on the actual size of the protein.
The most influencing factors are:
- Post-translational modifications
- Post-translational splits
- Isoforms and other variants
- Relative load
9.- How much sample should I load in the gel?
The amount will depend on the type of sample we handle:
- Cell lysates : the amount should be optimized based on the expression levels of the protein of interest in each case, but in general we can load between 20 and 30 ug of total protein per well.
- Purified protein : 10 to 100 ng of protein are usually loaded.
10.- Why can’t I detect my recombinant protein?
It could be because the recombinant protein that is expressed in the sample does not include the antigenic sequence recognized by the antibody that we are using, or in the case that the recombinant protein is expressed with a tag and it is very bulky or interferes with the antigenic sequence, could prevent its binding to the antibody.
In the case of working with recombinant proteins, it is always recommended to include an endogenous positive control in the Western Blot.
11.- Should I use milk or BSA as a blocking agent?
In general, BSA will give cleaner results since by containing fewer proteins, the probability of cross-reactions with the antibody is reduced.
However, in certain cases, blocking with milk will work better precisely because a greater variety of blocking proteins has the ability to block a greater range of different proteins.
12.- Why do so many bands appear in the Western Blot?
This may be due to several factors, including:
- The antibody is not specific enough for the target protein.
- Antigen degradation by proteolysis.
- Too much protein per lane.
- Overly sensitive detection system.
- Ineffective blocking.
- Antigen concentration too low.
13.- What is the difference between a Western Blot in reducing and non-reducing conditions?
To perform a Western Blot under reducing conditions, a reducing agent such as DTT or B-mercaptoethanol is added to the sample buffer to break the disulfide bridges, whereby the protein will be in its denatured form when the immunoassay is performed. .
14.- Should I use reducing or non-reducing conditions in my test?
Western Blots are usually performed under denaturing conditions. In any case, it is advisable to consult the technical sheet of the antibodies to ensure that they will work against the denatured protein.