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Western Blots

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 at http://www.bio-rad.com/LifeScience/pdf/Bulletin_2895.pdf.

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.

Sample Buffer:

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.

Destain Solution

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

Transfer Buffer

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.

Diluent Buffer

Dissolve 1.0 g non fat dry milk powder in 100 ml PBS-Tween Store in fridge for up to a week.

Procedure

  1. 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.
  2. 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/10007296.PDF
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7.  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
  8. For total protein staining, immerse the rinsed blot into the Amido black stain and shake gently for 10 minutes.  Destain with distilled water
  9. 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.
  10. 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.
  11. Wash the blot 3 times with PBS-Tween and once with PBS for 5 minutes each.
  12.  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.
  13. Wash the blot 3 times with PBS-Tween and once with PBS for 5 minutes each.
  14. 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

 

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References

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489

Ed Harlow & David Lane, Eds. Using Antibodies : A Laboratory Manual. Chapter 8. Cold Spring Harbor Laboratory Press, New York, 1999.

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