Agrisera Western Blot recommendations, troubleshooting and protocols
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Each antibody-antigen interaction has unique characteristics. A protocol giving good results for one antibody-antigen pair might found to be unsatisfactory for a second antibody even on the same sample. The interaction of antisera with protein epitopes in Western blot dependens 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. Polyclonal antibodies (serum or IgY-fractions from egg yolk) usually contain a number of different antibodies interacting independently with different epitopes on the target used for immunization. This number is limited for peptide-antigens but if recombinant or native proteins have been used for immunization the signal obtained has to be regarded as cumulative (=caused by several different types of antibodies present in the serum/IgY used).
To achieve reproducible results the usage of gloves, forceps, clean and detergent-free laboratory materials, reagents which did not pass expiry date, freshly prepared buffers with controlled pH, as well as keeping protocol volumes, dilutions, and times is of primary importance in addition to the steps discussed below. In general, incubation times should be as short as possible while antibody dilutions should be as high as possible.
Careful control of presence of the target protein in the starting material, its preparation and storage under non-degrading/aggregating conditions is essential. If the target protein is degraded or not present in sufficient amounts in the loaded sample, it can result in lack of signal detection. Epitope abundance can be enhanced under altered growth conditions, by selective tissue preparation, or fractionations of complex cellular extracts (i.e. organelle preparation). Use extraction method which provides good yields and consistent results. Agrisera has recently validated Precellys® device from Bertin Techonologies, as fullfilling such criteria. For work with organisms which posses robus cell walls like diatoms, some recommendations can be found here. Useful reference: "What is the total number of protein molecules per cell volume? A call to rethink some published values."Milo, 2013.
Sample quality is of crucial importance. As shown in the example below, in older samples of Arabidopsis thaliana total leaf preparations from year 2008 or 2006 protein of interest (in this example PsbB) has been degraded. M-molecular weight markers.
Secondary antibody used was Agrisera goat anti-rabbit HRP (1: 50 000) and ECL Advance detection system (GE Healthcare).
Protein samples older than 1 year should not be used if possible.
Run sample buffer (+ loading buffer) in one lane of your gel to check for contribution of your sample extraction buffer to the background signal.
Alternative preparation methods should be considered if possible.
Include positive and negative controls from the beginning: comparison of samples prepared from material containing no target (e.g. genetic 0-mutants) or higher amounts of the target (e.g. over-expression mutants) as well as cellular fractionation (e.g. preparation of various fractions of organells) will allow to control specificity of obtained signals.
Protein extraction protocols: seeds
(2) protein separation
The size-related identification of a protein-antibody interaction usually requires gel separation under fully denaturing conditions. Complete reduction of intra- and intermolecular S-S bridges ensures the accessibility of the epitope for interaction with antibodies detecting linear epitopes. In this cases, reducing agents have to be added to sufficient final concentrations to both, sample and running buffer. If antibodies recognize non-linear eptiopes they require conformational integrity of the target best provided in non-denaturating PAGE systems or immuno-histochemistry applications. Increasing protein loadings might elevate epitope abundance but most often also promote non-specific cross-reactions, higher background, and impaired separation.
Avoid protein loadings higher that 5-20 µg/lane for standard mini-gel systems.
Check separation with stained marker pattern or by Coomassie/Silver-staining of the gel. Mixing a pre-stained marker with a marker reacting with the secondary antibody (e.g. MagicMark, Invitrogen) will be an advantage for size-determination and can serve as a control for the visualization assay.
Some highly hydrophobic membrane proteins might require 2-8 M urea in the gels and sample buffer to be kept unfold completely during separation.
If DTT is used as reducing agent it should be freshly prepared.
If your polyacrylamide gel is not polymerazing wash glass plates carefully and make sure all fat is removed.
(3) protein transfer
The protein transfer on a suitable membrane (blotting) with protein-binding capacity (nitrocellulose, 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 times than smaller proteins. At low field strengths (<15 V/cm) the mobility of proteins out of the gel will be decreased and proteins will not be completely transferred from the gel. If field strength is too high (>35 V/cm) proteins might pass through the membrane without binding. The transfer time should be as short as possible to prevent proteins (especially lower molecular weight) from passing through the membrane. Place the second membrane and check how much signal is there after incubation with primary antibody compare to the first membrane.
- In gradient gels the porosity of the gel is matched with the size of the proteins what can be an advantage for efficient transfer.
- Check efficiency of a transfer by post-transfer staining of the gel (e.g. Coomassie or Silver) or the filter (Ponceau, reversible).
- Drying of a membrane (air dry between clean sheets of filter-paper) can improve immobilization of the protein.
- for some antibodies nitrocellulose membrane might work better compare to PVDF and opposite way around.
- It is crucial that blotting equipment is well rinsed with destiled water after each use and is kept away from contaminating detergents. Foam pads should be cleaned and thoroughly washed. When your pads aquire colours (from plant material) and/or loose tension it is time to change them.
- Do not reuse transfer buffers.
- The presence of SDS (0.01 to 0.02 %) in transfer buffer will increase the mobility of proteins (especially large proteins) out of the gel and provide a negative charge to the protein, which will help to maintain it in the soluble state. At the same time, SDS will reduce protein binding to the membrane (especially nitrocellulose) due to decreased hydrophobicity of the protein.
- The presence of alcohol in the transfer buffer will decrease protein mobility out of the gel. It will also reduce pore size of the gel, while it will improve binding to nitrocellulose as it removes SDS from proteins and increases their hydrophobicity. Higher molecular weight proteins might not be transferred completely if methanol is present in the transfer buffer. Change membrane to nitrocellulose, omit methanol from transfer buffer, while adding SDS and increasing field strength.
- The thickness of the gel is affecting protein mobility out of a gel. Thicker gels allow higher loading but lower molecular weight proteins might transfer less efficiently.
- Hydrophobic proteins might migrate further in the gel than they "should" based on their molecular weight. This is because hydrophobic proteins bind more SDS per amino acid, skewing the chrge to mass ration (more negative charge, therefore they migrate faster).
To minimize background staining due to non-specific membrane-binding of the antibody tested, remaining surface of the membrane has to be saturated (“blocked”) with proteins which are not reacting with the antibody. Commonly used are low-fat dry milk powder, casein, IgGs (at 5-10 % but not from a species you have your antibodies derived from) or BSA, at 2-5% w/v. The shortest possible blocking-time should be determined experimentally for your system. Increasing the blocking-times unnecessarily (e.g. overnight) or incubation at low temperatures might lead to aggregation of blocking protein on the membrane and by this compromising later antibody-epitope interactions. If low background is obtained after 30-60 min of blocking any longer blocking will not improve your results. It should be determined experimentally if adding or omitting the blocking reagent in the subsequent antibody incubation steps is improving the results. Usually the buffer and the detergent concentration used should not abolish interaction of the blocking-protein with the membrane. However, accessibility of a target protein for primary antibody can be increased by a short wash using a wash buffer without blocking protein, performed before incubation with primary antibody. Each antibody-antigen pair is unique and therefore choice of blocking should be emprirically tested.
As some IgY antibodies might recognize milk proteins, BSA might give lower background than milk powder in such cases.
Do not use BSA as blocking agent if BSA-coupled peptides were used for immunization. BSA should be of high purity, IgG-free not to interfere with the assay.
As milk contains biotin, the use of milk-power for blocking is incompatible with avidin/streptavidin systems.
If serum is used for blocking it should be considered if it can be excluded that the animal from which blocking serum has been obtained may have been exposed to and 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. Purified immunoglobulins are higher quality blocking reagents.
As a protein-free protein alternative you may block for 1 hr at RT with 0,2-0,5 % Tween-20 in PBS followed by incubation with the primary antibodies diluted in 0,25 % Tween-20 in PBS for 1 hr at RT.
(5) primary antibody
The primary antibody is the major determinant of the specificity of the target-recognition. The interaction with the primary epitope should be in favour over any cross-reactivity with other similar epitopes. Commonly used dilutions are between 1:500 to 1:20 000, depending upon the reactivity of the antibody 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) 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.
Lyophilized primary antibodies can be incubated for 2-4 h at 4º C after reconstitution prior to use.
To diminish unspecific cross-reactions you may pre-adsorb the primary antibody (overnight, 4º C) with tissue extract which lacks your protein of interest (you can use a transferred membrane where the band/area containing your target protein has been cut out). In case of increased background signals, incubate your membrane before any protein is transferred in saturated PBS-milk followed by protein transfer.
Antibodies against carrier proteins (KLH, BSA or others) might recognize epitopes of other proteins present in the extract. Those anti-carrier antibodies can be removed by (1) incubating serum/IgY sample with a carrier protein in solution (0.1 % (w/v) or spotted on PVDF membrane or (2) passing serum/IgY sample through the column with bound carrier protein (use flow through fraction in further experiments).
Primary antibodies stored (at 4°C or -20°C) in solution with blocking reagent for further use might lose their activity as well as a certain portion of antibodies will be depleted from such solution. This might result in weaker signal for every next blot.
Lots of small spots on a mebrane obtained after development of a western blot can be a results of fat present in a given serum. To improve such blots, use milk based blocking reagents as well as increase Tween concentration in your bufffers.
(6) secondary antibody
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. Thus, 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”). Usually secondary antibodies are used at dilutions of 1:20 000-1:500 000 depending on the sensitivity of the visualization method (e.g. enhanced chemi-luminenscense, ECL or alkaline phosphatase, AP). 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 (see example for trouble shooting).
If an Ig-reactive marker has been used the signals obtained from the marker can serve as a control of the function of the secondary antibody as well as the subsequent visualization.
Sensitivity of secondary antibodies can differ between various manufactures
10 μg of mitochondrial fraction from Arabidopsis thaliana (1,3) and Arabidopsis thaliana leaf extract (2,4) were separated on 10% gel and blotted on nitrocellulose membrane using wet transfer (0.22% CAPS, pH 11). Filters where blocked (1.5h) in 5% milk in TBST (1X TBS, 0,1% Tween 20), incubated with 1: 1000 anti-COXII antibodies (2h in TBST) followed by incubation with 1: 10 000 secondary anti-rabbit (1h) HRP-coupled antibodies from Agrisera (left panel) and other manufacture (right panel) and visualized with standard ECL on Kodak autoradiography film for 5 s. Antibody in left panel detects target protein also in total cell extract (2) and can be used in higher dilution than applied 1: 10 000. Agrisera goat anti-rabbit HRP conjugated antibody (AS09 602) can be used at following dilutions: 1: 50 000 -1: 90 000 (ELISA), 1 : 75 000 with enhanced ECL and 1: 25 000 with regular ECL (WB), 1: 500 -1: 5000 (IHC).
After all steps (4-6) excess off blocking-protein, primary or secondary antibody has to be diminished by washing. Usually same buffers as used for the preceeding steps are used. Typically they will include a detergent, such as 0.05 % up to 0.5 % of Tween20. Blocking solution in 1:10 dilution can be added to the wash buffer. Presence of the blocking agent with detergent might help to minimize background in the assay by preventing elution of the blocking protein from the membrnae. In some protocols washing steps include subsequent changes of the washing volumes with omitting the detergent in the last step. For reasons of reproducibility it is recommended to keep volumes and times constant. The intensity of washing steps can be elevated by (a) increased times and volumes, (b) additional changes of buffer, (c) higher detergent concentrations, (4) use of stronger detergents (e.g. SDS instead of Tween-20, use high-purity detergents as they might contain high amounts of peroxides and contribute to increased background). Pre-diluted stocks of detergents can be prone to microbial growth, which in turn can contribute to increased background noise.
Enzymatic detection systems are most popular, which employ usage of secondary antibodies conjugated with Alkaline phosphatase (AP or ALP) or horseradish peroxidase (HRP). In our hands alkaline phosphatase detection system is at least 10x less sensitive comapre to chemiluminescence based system (like ECL). Therefore using ALP is not recommened when working with proteins of low expression levels in the tissue. In such cases more sensitive detection systems should be employed (like TMA-6 from Lumigen). Some laboratories have a possibility to use fluorophore-conjugated antibodies (like DyLight®) and in such cases there is no substrate development step in the assay. The protocol is shorter and due to advances of digital imaging few fluorophores can be used in the same assay.
(9) membrane storagemembranes (nitrocellulose or PVDF) can be stored after western blot has been completed for up to 6 months in room temperature between sheets of Clinex.
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