Each antibody-antigen interaction has unique characteristics. Interaction of antisera with protein epitopes in western blotting depends upon a number of factors, all contributing to the final signal/noise ratio. Knowledge and control of those factors allows for modifications and optimization of the procedure.
Prepare the membrane
For size-related identification of an interaction between protein and antibody a gel separation under fully denatured conditions is required. Complete reduction of intra- and intermolecular S-S bridges ensures the epitope is accessible for interaction with antibodies, especially those capable of detecting linear epitopes. In this case, reducing agents have to be added during sample preparation to achieve a sufficient final concentration of both the sample and the running buffer. If antibodies recognize non-linear epitopes, they require conformational integrity of the target, which may be provided in non-denaturing PAGE systems or immuno-histochemistry applications. Increasing the protein load might elevate epitope abundance, but may also promote non-specific cross-reactions, higher background, and an impaired separation. Users should avoid loading protein higher than 5 to 20 µg per lane for a standard lane.
Check your separation with a stained marker pattern or by Coomassie/Silver-staining of the gel. Mixing a pre-stained marker with a marker that reacts with the secondary antibody provides an advantage for size-determination and can serve as a control for the visualization assay.
Highly hydrophobic membrane proteins may require 2 – 8 M urea in the gels and sample buffer to be kept unfolded during separation.
If DTT is used as the reducing agent, it should be freshly prepared. If your polyacrylamide gel is not polymerizing, wash the glass plates carefully and make sure all grease is removed.
About the protein transfer
Blotting, or transfer of the protein to a suitable membrane with protein-binding capacity, such as nitrocellulose or PVDF, is highly dependent on the biochemical properties of the target protein. Proteins of higher molecular weight (apparent molecular masses of >100 kDa) require longer blotting time than smaller proteins, and at low field strengths (<15 V/cm), a longer transfer time is required.
Since protein retention on a membrane depends on the pore size and electrostatic properties of the membrane, the best results for small proteins (less than 10 kD) are achieved using nitrocellulose membranes with smaller pore sizes (less than 20 um). The quality of transfer could be affected by staining the membrane with Ponceau S.
Air drying of PVDF membrane between clean sheets of filter-paper can improve immobilization of the protein. For some antibodies, nitrocellulose membrane might work better than PVDF, and the reverse is true for other antibodies. Blotting equipment must be well rinsed with distilled water after each use and kept away from contaminating detergents.
It is advisable to not reuse transfer buffers. The presence of SDS (0.01 to 0.02 %) in the transfer buffer increases the mobility of proteins (especially large proteins) out of the gel and will also provide a negative charge, which helps to maintain it in a soluble state. SDS reduces hydrophobicity of the protein and its binding ability to the membrane (especially nitrocellulose).
Presence of alcohol in the transfer buffer decreases protein mobility out of the gel. It will also reduce gel’s pore size, while it will improve binding to nitrocellulose as it removes SDS from proteins and increases hydrophobicity. If higher molecular weight proteins are not completely transferred due to the presence of methanol in the transfer buffer, change the membrane to nitrocellulose, omit methanol from transfer buffer, and add SDS to increase the field strength.
Some strongly hydrophobic proteins might migrate further in the gel than they expected based on their molecular weight because they bind more SDS per amino acid thus skewing the charge-to-mass ratio. Nitrocellulose or PVDF membranes may be stored after blotting for several months at room temperature or in a refrigerator between sheets of tissue paper (like Kleenex or Kim Wipes) to keep it protected from dust.
Upon successfully performing all the steps for protein transfer, consider using automation for blocking, incubation and washing steps to minimize potential errors that may cause inconsistencies and variations in results. BlotCycler is simple, effective and well worth the investment, especially considering its ability to operate unattended.
Blocking for success
Membranes are blocked with proteins such as low-fat dry milk powder, casein, immunoglobulin G (at 5-10% but not from a species you have your antibodies derived from) or BSA, at 2-5% w/v. Proper blocking reagents are non-reactive with the antibody used in the assay. The blocking-time should be determined by trial and error for each experiment, but generally should be about 1 hour. Applying centrifugation to the blocking agent prior to use prevents aggregation to the membrane, in particular if blocking is done at low temperature. If low background occurs after 30 to 60 minutes, continued blocking will not improve your results.
It should be determined experimentally if adding or omitting the blocking reagent in subsequent antibody incubation steps yields an improvement on the results. Normal buffer and detergent concentrations should not reduce the efficiency of blocking; however, accessibility of a target protein for primary antibody is increased by a post blocking wash (1-3 times). Each antibody-antigen pair is unique and therefore the blocking protocol should be empirically tested. As milk contains biotin, the use of milk-power for blocking is incompatible with avidin/streptavidin systems. If serum is used for blocking, care should be taken to ensure the animal has not been exposed to (or developed antibodies to) the antigen in question. If this is the case, they may bind to the antigen and prevent binding of the primary antibody. Consider using purified immunoglobulins as they are higher quality blocking reagents. As a protein-free protein alternative you may block for 1 hour at RT with 0.5 % Tween-20 in PBS followed by incubation with the primary antibodies diluted in 0.1 % Tween-20 in PBS for 1 hour at RT.
Primary antibody incubation
The primary antibody is the major determinant of the specificity for the target-recognition. The interaction with the primary epitope should dominate any cross-reactivity with other epitopes. Commonly used dilutions are between 1:500 to 1:20,000, depending upon the reactivity of the antibody and the detection system used. To check for specificity of the target recognition you can use (1) another antibody against your target which will bind to other epitopes on a target protein, (2) run control samples free or depleted of target-proteins, or (3) perform a peptide competition assay (for anti-peptide antibody). If available, a second antibody known to react with the sample can be used as a control of subsequent assay steps on a parallel filter in the same experiment.
To reduce unspecific cross-reactions you may pre-adsorb the primary antibody (overnight, 4º C) with tissue extract that lacks your protein of interest (you can use a transferred membrane where the band/area containing your target protein has been cut out). Primary antibodies stored (at 4°C or -20°C) in solution with blocking reagent often lose their reactivity. Antibody dilutions should be stored in buffer only, not buffer plus the blocking mixture. Lots of small spots visible on a membrane during development can be due to fat in a given serum. To improve such blots, use milk based blocking reagents as well as increasing Tween concentration in your buffers.
Washing after primary antibodies
To achieve quality results, it is important that the washing step after introducing the primary antibodies are performed consistently, preferably using an automated system. Washing can be done at RT or 4C; however, multiple observations show that washing at 4C increases signal intensity.
Secondary antibody incubation
The secondary antibody has to be reactive against the primary antibody (e.g. use anti-rabbit to detect primary antibodies raised in a rabbit) and usually is coupled to an enzyme or dye that allows subsequent visualization. Any non-target binding of the secondary antibody will result in background (if bound to the membrane due to insufficient blocking) or false-positive recognition of non-target proteins present on the filter (“cross-reactions”). It is important that the optimal dilution of the secondary antibody has to be determined experimentally for the detection system used. Different secondary antibodies may even result in different recognition patterns when applied to the same sample. To check for contribution of the secondary antibody for the result you may cut a suitable are of your filter and run it as a parallel control where the primary antibody is omitted.
Washing after secondary antibodies
The washing after secondary antibodies is less important than washing after primary antibodies, but also should be performed under standardized conditions to control the volume of washing solution and the time of washing. It is also better to perform washing at 4C. The intensity of washing steps can be elevated by increased the number of times of buffer changes but not the time of incubation. It is good practice to verify that stocks of buffers (including wash buffer) do not contain microbial growth (visible as cloudiness that settles), as this can contribute to increased background noise.
Enzymatic detection systems are most popular – they typically use secondary antibodies conjugated with Alkaline phosphatase (AP or ALP) or horseradish peroxidase (HRP). Use of fluorescent secondary antibodies actually provides better quantitative assay results.
Serious science requires standardization
As popular as western blot assays are, the number of processing steps that have to be performed precisely, time after time, strongly suggests the need for an automated system. BlotCycler is extremely helpful for closely following assay protocols, maintaining uniform volumes and dilutions, and precisely controlling incubation times. If you are serious about achieving western blot reproducibility, consider employing an automated system, such as BlotCycler. For more on BlotCycler, visit this page: www.blotcycler.com.