Published: Thursday, 08 May 2014 21:19
Described here is a Western Blot protocol, sometimes called Immunoblotting, a procedure which will identify your immunizing antigen (via a blotted SDS Polyacrylamide gel) using your primary Chicken IgY antibody as a probe. This is the protocol that we use at Gallus Immunotech. There are many variations and a more detailed description of the procedure can be found here.
Reagents and Buffers:
Polyacrylamide Gel Electrophoresis Buffers:
1 M Tris Buffer, pH 6.8
Dissolve 12.1 grams Tris Base in 50 ml distilled water. Adjust the pH to 6.8 with concentrated HCl.
QC to volume of 100 ml. Store in fridge.
2.0 ml Glycerol
0.2 g Sodium Dodecyl Sulphate (SDS)
0.1 g Bromophenol Blue
0.6 ml 1 M Tris buffer, pH 6.8
1.0 ml 1 M Dithiothreitol (DTT) (added fresh from frozen stock), if you want a reduced protein profile
QC to volume of 10 ml with distilled water and mix thoroughly. Sample buffer without DTT may be refrigerated.
10X SDS Polyacrylamide Gel Running Buffer
30.3 g Tris base
144.0 g Glycine
10.0 g Sodium Dodecyl Sulphate (SDS)
QC to volume of 1 L with distilled water and mix thoroughly. Store in fridge.
Coomassie Blue Gel Stain (0.1%)
0.25 g Coomassie Briliant Blue R-250 powde
100 ml Methanol
25 ml Glacial Acetic Acid
QC to volume of 250 ml with distilled water and filter (paper, Whatman #1). Label and store at room temperature.
150 ml Methanol
50 ml Acetic Acid
QC to volume of 500 ml with distilled water. Label and store at room temperature.
Western Blot Buffers
3.0 g Tris base
14.4 g Glycine
200 ml Methanol
QC to volume of 1 L with distilled water. Label and store in refrigerator.
Amido Black Stain
40 ml Methanol
10 ml Acetic Acid
0.1 g Amido Black Stain
QC to volume of 100 ml with distilled water. Label and store at room temperature.
1 M Potassium Phosphate Buffer, pH 7.3
660 ml 1 M K2HPO4
330 ml 1 M KH2PO4
Mix thoroughly. Check pH. Adjust if necessary (to lower pH, add more 1M KH2PO4; to raise pH, add more 1M K2HPO4). Store in fridge.
Phosphate Buffered Saline (PBS)
30 ml 5 M NaCl
10 ml 1 M Potassium Phosphate buffer, pH 7.3
QC to volume of 1 L wtih distilled water and mix thoroughly. Store in fridge.
Phosphate Buffered Saline-Tween, 0.05% (PBS-Tween)
Add 0.5 mL Tween 20 to 1 L PBS.
Blocking Buffer (5% milk in PBS)
Dissolve 5 g non fat dry milk powder in 100 ml PBS. Store in fridge for up to a week.
Dissolve 1.0 g non fat dry milk powder in 100 ml PBS-Tween Store in fridge for up to a week.
- Prepare your experiment by noting what protein samples are going in what wells on your gel, the % of acrylamide of your gel, whether it’s a gradient gel and the amount of protein you are adding per well. For a mini gel, it is recommended to not add more than 5 ug and if the protein sample consists of few proteins, it is advisable to add even less protein. You may want to include a well with pre-stained molecular weight marker and by running a duplicate gel, you can stain one gel (or transferred gel) for protein and immunoblot the second.
- Run your polyacrylamide gel electrophoresis according to the instructions of the manufacturer. We use BioRad’s Mini Protean System: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006157B.pdf
- Once it is finished running, remove the gel and equilibrate immediately in Transfer Buffer along with nitrocellulose (or polyvinylidene difluoride (PVDF) membrane), 2 absorbent filter papers and 2 fibre support pads for at least 30 minutes, ensuring that the gels and membranes are fully immersed in the buffer.
- Meanwhile, place the duplicate gel in Coomassie blue for an hour shaking gently. Destain overnight shaking gently. You may also choose to transfer both gels and protein stain the duplicate blot with Amido Black.
- To assemble the sandwich for protein transfer onto the membrane, place the open transfer cassette in a shallow vessel containing Transfer buffer. In this order, place fibre support pad, filter paper, equilibrated gel, membrane (making sure there are no air bubbles), filter paper and fibre pad. Roll carefully with the side of a beaker to remove air bubbles and ensure maximal contact. Carefully close the cassette.
- Place the cassette in the transfer tank containing transfer buffer with the membrane closest to the red (positive) electrode (anode). When the cassette is in the tank, the buffer should completely cover the cassette. If possible, add ice pack to prevent overheating. Hook up to power pack and transfer for 60 minutes at 100 volts or overnight at 25 volts.
- Remove the blot from the cassette and rinse thoroughly with water. Divide the blot into the sections that will be immunostained or stained for total protein (Amido Black). Mark the membrane pieces by notching one corner
- For total protein staining, immerse the rinsed blot into the Amido black stain and shake gently for 10 minutes. Destain with distilled water
- To immunostain your blot, first block the protein binding sites by transferring the blot to Blocking Buffer, shaking gently for 1 hour at room temperature or in the refrigerator overnight.
- After blocking, rinse the blot twice in PBS for five minutes each. In a shallow tray, add the primary antibody diluted in Diluent Buffer. The recommended antibody concentration should be in the range of 1 to 50 ug/ml. Incubate at least 1 hr at room temperature, gently shaking. Sensitivity may increase with a longer (overnight) incubation.
- Wash the blot 3 times with PBS-Tween and once with PBS for 5 minutes each.
- Now, prepare the labeled anti-IgY secondary antibody. We use our Horseradish Peroxidase (HRP)-labeled Donkey anti-IgY (Cat. # DAIgY-HRP) at a 1:15000 dilution (0.07 ug/ml). A ball park figure for diluting secondary conjugated antibodies is between 0.5 and 5 ug/ml. Incubate for 1 hr at room temperature, gently shaking.
- Wash the blot 3 times with PBS-Tween and once with PBS for 5 minutes each.
- Your immunoblot is ready to be developed now. The enzyme (HRP)-conjugated secondary antibody in the presence of its substrate (we use 3,3,5,5-Tetramethylbenzidine or TMB) catalyzes the conversion of that substrate to a coloured product. These substrates are available commercially, for example: https://www.lifetechnologies.com/order/catalog/product/37574
Other IgY Resources:
Published: Thursday, 08 May 2014 21:11
Affinity Purification of IgY
The purified egg yolk IgY fraction, as well as containing IgY directed against the immunizing antigen, contains all the other antibody specificities that were present in the animal\'s circulation at the time the eggs were formed. The IgY fraction, depending on the purification method, is approximately 85-90% pure (as determined by SDS polyacrylamide gel electrophoresis).
The specific activity (percentage of immunoglobulin Y that is directed against your antigen) of this IgY fraction depends on the immunogenicity of your antigen and the genetic make-up of the immunized hen. It is reasonable to expect between 2 and 10% specific activity of IgY from a chicken immunized with a protein greater than 50 kilodaltons. Expect less than 1% specific activity to a peptide from a hen immunized with a peptide-KLH conjugate (there will be a much higher specific activity directed to the carrier KLH portion of the immunogen). For many applications, this non-affinity purified antibodiy fraction will work just fine and it is unnecessary to further purify your IgY. In fact, further purification may result in the loss of your highest avidity antibodies.
However, sometimes, it is necessary to “affinity purify” your IgY fraction, ie to remove the IgY antibodies that are not antigen-specific, leaving only the IgY that recognizes the immunizing antigen. You might want to perform this extra step, especially if you’re finding that there is “non-specific” or cross-reactivity occurring in your immunoassays. To perform affinity purification, it is first necessary to bind your immunizing protein to a solid phase.
Preparing your "Immunizing Antigen" Solid Phase
If enough of your immunizing protein antigen is available, the most common solid phase support is an agarose bead. Per 1 ml agarose bead, the amount of protein antigen (or peptide) required for an efficient column would be approximately 10 mg. (If immunizing protein is limited in availability, please see the end of this section for an alternative analytical method, using nitrocellulose as your solid phase. ) Agarose resins, already chemically activated to bind to your protein or peptide immunogen via a free amino or sulfhydryl group, are available commercially. Some examples are listed below:
For the binding of your antigen to the activated resin, follow the instructions of the resin’s manufacturer, making sure that you measure the protein concentration of your sample (optical densitiy at 280 nm) before binding and after binding, to ensure that the binding to the agarose has taken place.
Applying your IgY Sample to the Affinity Column
Measure the concentration of your IgY fraction (purified using one of the procedures outlined in Table I), using the extinction coefficient 1.36. Make a 1:10 dilution of your IgY and measure the absorbance at 280 nm. Calculate the IgY concentration by using the following formula:
IgY concentration (mg/ml) = Absorbance (280 nm) x 10 (IgY dilution factor 1.36 (extinction coefficient)
Dilute your IgY fraction to approximately 1 mg IgY / ml PBS. Apply the diluted IgY to your antigen column (use about 80 mg IgY per ml antigen-bound agarose), at a very slow drip, using a peristaltic pump. When the sample has finished, apply PBS to the column, until the PBS coming through the column has an absorbance (at 280 nm) of 0. Now apply a PBS solution to which NaCl (0.5 M) has been added, again until the absorbance (280 nm) is 0. Now, it’s time to elute the antigen-specific IgY.
Eluting your Antigen-Specific IgY
The most common method to dissociate your IgY antibody from its ligand is by an extreme change in the pH. The antigen-specific IgY is then eluted off, into a neutralizing buffer. Other elution buffers may contain high molarity salts and/or chaotropic agents. You may make up your own buffers or purchase them. Pierce Biotechnology has a good selection of buffers that you may need. Eluting your antigen-specific IgY may require some experimentation to determine the optimal elution buffer. The following method will work in most circumstances:
Apply at a slow rate, a 0.1 M glycine buffer, between pH 2.0 and 3.0 (this may require testing for optimization; try pH 2.3 to start), making sure that the eluted IgY is dripping into a neutralizing buffer (0.2 M sodium phosphate buffer, pH 8.0). Continue until the IgY eluate absorbance is 0. The ratio of neutralizing buffer: elution buffer should be about 1:4. As soon as the column eluate has a 0 absorbance (280 nm), neutralize the column by running PBS through, until the pH of the eluate is neutral.
Now, you want to determine whether you still have specific IgY remaining on your column, that would be eluted in an alkaline environment. Apply at a slow rate, 0.1 M triethylamine or triethanolamine, pH 11 -11.5, making sure that the eluted IgY is dripping into a neutralizing buffer (1.0 M sodium phosphate buffer, pH 5.0). Continue until the IgY eluate absorbance is 0. The ratio of neutralizing buffer: elution buffer should be about 1:25-30. As soon as the column eluate has a 0 absorbance (280 nm), neutralize the column by running PBS through, until the pH of the eluate is neutral. If no IgY was eluted at the alkaline pH, it is not necessary to perform alkaline elution on the next run.
Your column is now ready for another affinity purification procedure. To store your column, add 0.1% sodium azide and store in refrigerator.
Modification of Affinity Purification Procedure, when immunizing antigen is limiting
If only analytical quantities of affinity-purified IgY are required and antigen is limiting, you may want to blot your antigen on nitrocellulose and let the blot dry (or alternatively, transfer onto nitrocellulose from an SDS gel). Incubate a small amount of your diluted IgY fraction with the antigen blot and place on a shaker for an hour. Wash blot thoroughly with a 0.05% Tween 20 PBS solution until the washing solution has an absorbance of 0 (280 nm). Add your elution buffer (as described above), shake briefly and quickly neutralize your eluate (as described above). Wash your nitrocellulose blot in PBS until eluate is neutral and store in 0.1% sodium azide in refrigerator.
Concentrating your Antigen-Specific IgY
You may want to concentrate your affinity purified IgY – it will be more stable and take up less room in the fridge. If you decide to store it in a diluted form (less than 1 mg/ml), consider adding BSA, to a concentration of 1%.
Unlike mammalian IgG, IgY does not bind to protein A or protein G, so concentration of your affinity-purified IgY using protein A or protein G columns is not an option. Ultrafiltration and ultra-centrifugal filtration are effective, albeit expensive procedures that will concentrate your specific IgY. A cheaper alternative is to precipitate the IgY in your sample with 20% ammonium sulphate. After 1 hour incubation in the fridge, spin out your IgY and resuspend in PBS. Add sodium azide to a concentration of 0.1 percent and store at 4°C.
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