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Protein separation by SDS-PAGE and staining procedures

Analysis of proteins on the membranes

Storing and testing antibodies

Purification of antibodies



Protein separation by SDS-PAGE and staining procedures

Denaturing gel-electrophoresis (according to Laemmli 1970)

10 x Laemmli running buffer:0.25 MTris-HCl, pH 8.5
1.92 Mglycine
1%SDS
   
4% Laemmli sample buffer:0.25 MTris-HCl, pH 6.8
8%SDS
40%glycerol
20%ß-mercapthoethanol
0.016%Bromphenol Blue
   
Separating gel:0.375 MTris-HCl, pH 8.8
12%acrylamide (acrylamide: bis-acrylamide 29 : 1)
0.1%SDS
0.06%APS
0.006%TEMED
   
Stacking gel:0.125 MTris-HCl, pH 6.8
5%acrylamide
0.1%SDS
0.08%APS
0.008%TEMED


  1. If using a commercial apparatus, assemble the gel plates following manufacturers instructions.

  2. Prepare the acrylamide solution for separating gel. Add TEMED before pouring the gel. The gel will polymerise within 5 10 min after addition of TEMED.

  3. Pour the acrylamide solution between the plates. The meniscus of the acrylamide solution should be far enough below the top of the notched plate to allow for the length of the teeth on the comb plus 1 cm. Carefully overlay the acrylamide solution with isobutanol or water. For gels made with acrylamide concentrations lower than 8% use water; for gels of 10% or greater use isobutanol. Place the gel in a vertical position at room temperature.

  4. After the gel has set (about 20 30 min), pour off the overlay and wash the top of the separating gel several times with distilled water. Drain well or dry with the edge of a paper towel.

  5. Prepare the acrylamide solution for the stacking gel. Pour the solution for the stack directly onto the polymerized separating gel. Place the appropriate comb into the gel solution, being careful not to trap any bubbles. To clean the comb, wash with H2O and ethanol. Place the gel in a vertical position at room temperature. The stacking gel will set in approximately 10 min.

  6. Heat samples in 1 - 2 x Laemmli sample buffer at 80C for 10 min.

  7. After the stacking gel has set, carefully remove the comb. Wash the wells immediately with distilled water to remove unpolymerised acrylamide. Straighten the teeth of the stacking gel, if necessary, and place in an appropriate gel box with running buffer in the bottom reservoir. Add running buffer to top reservoir.

  8. Load samples into the bottom of the wells. The samples can be conveniently loaded using a microliter syringe or a micropipette.

  9. Start electrophoresis at 100 125 V. After the dye front has moved into the separating gel, increase to 200 V.

  10. When the dye front reaches the bottom of the gel, turn off the power pack. Remove the gel plates and gently pry the plates apart. Use a spatula or similar tool to separate the plates (not at an ear).

    The gel is ready now for fixing, staining, transfer, fluorography or autoradiography.


Reference:
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 685



Coomassie Brilliant Blue staining of polyacrylamide gels

Staining solution:40%ethanol
5%acetic acid
0.2% (w/v)Coomassie Brilliant Blue R-250
   
Destaining solution:30%ethanol
7%acetic acid


  1. Incubate the gel in a staining solution for 30 min under constant shaking.

  2. Destain the gel in several changes of destaining solution (3 times 30 min) until the blue-stained protein bands will be well visible on the transparent gel background.




Silver nitrate staining of polyacrylamide gels

Fixation solution:50%ethanol (or methanol)
12%acetic acid
0.05%37% formaldehyde
   
Thiosulfate solution:0.02% (w/v)natrium thiosulfate
   
Silver solution:0.2% (w/v)silver nitrate
0.075%37% formaldehyde
   
Developing solution:6% (w/v)natrium carbonate
0.05%37% formaldehyde
0.0004%natrium thiosulfate
   
Stopping solution:50%ethanol (or methanol)
12%acetic acid


  1. Incubate the gel at least for 1 h in fixation solution.

  2. Wash the gel three times for 20 min in 50% ethanol (or methanol) in order to remove acetic acid.

  3. Soak the gel for 1 min in thiosulfate solution.

  4. Rinse the gel three times for 20 30 sec with water and incubate it in a silver solution for 20 min.

  5. Rinse the gel twice for 20 30 sec. with water and incubate it in the developing solution until protein bands reach the desired intensity (yellow till brown on the transparent background).

  6. Stop the gel staining with stopping solution.


Note: the incubation times are given for the gel of 1 mm thick. If you are using thicker gel (1.5 or 2 mm) you have to increase the incubation time proportionally.



Imidazol staining of polyacrylamide gels

Imidazol staining which does not fix proteins irreversibly can be performed prior to protein transfer onto the nitrocellulose or PVDF membrane.

Buffer 1:0.2 M imidazol
  
Buffer 2:0.3 M zink sulphate


  1. Incubate the gel in buffer 1 under constant shaking for 10 min.

  2. Exchange buffer 1 with buffer 2. Incubate the gel in buffer 2 till proteins will be visualised as transparent bands on a white milky background of the gel.

  3. Stop the gel staining by placing the gel into the water. For protein transfer onto the membrane the gel can be destained in a blotting buffer.


Note: Imidazol staining is not compatible with silver staining. Coomassie Brilliant Blue staining can be used immediately after imidazol staining (without destaining).



Analysis of proteins on the membranes

Wet-blot transfer of proteins onto nitrocellulose or PVDF membranes

10 x blotting buffer:1.5 M glycine
0.2 M Tris-HCl, pH 8.0


  1. Incubate nitrocellulose membrane and polyacrylamide gels for 10 min in 1 x blotting buffer prior to transfer. Pre-wet the PVDF membrane in methanol, ethanol, or isopropanol.

  2. Soak two sheets of Whatman paper of the gel size in a blotting buffer and place it on the cathode plastic plate.

  3. Place the gel on the top of the paper and cover it with nitrocellulose or PVDF membrane.

  4. Cover the membrane with two layers of Whatman paper soaked in 1 x blotting buffer and an anode plastic plate.

  5. Remove air bubbles with a blotting roller or glass tube.

  6. Perform the transfer for 2 3 h at constant 45V.

  7. Fix the proteins on the membrane by its backing for 10 min at 80C.




Semi-dry transfer of proteins onto nitrocellulose or PVDF membranes

Anode buffer 1:0.3 M Tris-HCl, pH 8.0
  
Anode buffer 2:0.025 M Tris-HCl, pH 8.0
  
Cathode buffer:0.04 M ε-aminocaproic acid


  1. Soak three pieces of 2.5 mm thick blotting filter paper in a cathode buffer and place it on the cathode plate.

  2. Place the pre-soaked in anode buffer 2 polyacrylamide gel on the top of the filter paper stack and remove any air bubbles with the blotting roller or glass tube.

  3. Soak the transfer membrane in the anode buffer 2 and place it on the gel.

  4. Soak three pieces of blotting filter paper in the anode buffer 1 and place it on the top of the membrane. Remove the air bubbles with the blotting roller or glass tube.

  5. Place the anode plate on the stack without disturbing the blot sandwich. Perform transfer for 2 h at 0.8 mA per cm2 of the gel.


Note: If you are using PVDF membrane do not forget to pre-wet it for 30 sec. in methanol, ethanol, or isopropanol. Briefly rinse the membrane in deionised water.



Staining the blot with Ponceau S

10 x Ponceau S solution:0.2% Ponceau S
3% trichloroacetic acid
3% sulfosalicylic acid
  
10 x PBS:0.75 M NaCl
30.0 mM KCl
45.0 mM Na2HPO4 x 12 H2O
15.0 mM KH2PO4


  1. Incubate the nitrocellulose membrane in 1 x Ponceau S solution for 30 min.

  2. Transfer the nitrocellulose sheet to 1 x PBS (or water) and rinse for 1 2 min with several changes of 1 x PBS (or water).

  3. Mark position of transfer and molecular weight markers with pencil or indelible pen. The nitrocellulose membrane is now ready for blocking.


Note: Ponceau S staining is rapid but not permanent. Because the binding is reversible, the stain is compatible with most antigen visualization techniques.



Staining the blot with India ink

Ink solution:100 l India ink (or equivalent one) in 100 ml of 03% Tween 20/1 x PBS
10 x PBS:0.75 M NaCl
30.0 mM KCl
45.0 mM Na2HPO4 x 12 H2O
15.0 mM KH2PO4


  1. Wash blot in 04% Tween 20/1 x PBS, with two changes of 100 ml at 5 min each.

  2. Place blot in ink solution and incubate at room temperature for 15 min to 18 h. Longer incubations increase sensitivity.

  3. Destain by washing the blot in multiple changes of 1 x PBS. The nitrocellulose membrane is ready for staining.


Note: The staining depends on the preferential adherence of the colloidal carbon particles in India ink to the immobilized protein on the filter. It is essential that the correct India ink be used. Pelican Fount India drawing ink (original recipe, black) or equivalent is suitable.



Western blot analysis using horseradish peroxidase-conjugated antibodies

10 x PBS:0.75 M NaCl
30.0 mM KCl
45.0 mM Na2HPO4 x 12 H2O
15.0 mM KH2PO4
  
Blocking buffer:1 x PBS
5% low-fat dry milk
1% Tween 20
  
Washing buffer:1 x PBS
1% Tween 20
  
Stock solution 1:0.25 M luminol (in DMSO)
  
Stock solution 2:0.09 M p-coumaric acid (in DMSO)
  
Developing solution 1:2.5 mM luminol
0.4 mM p-coumaric acid
0.1 M Tris-HCl, pH 8.5
  
Developing solution 2:5.4 mM H2O2
0.1 M Tris-HCl, pH 8.5


  1. Transfer the proteins from polyacrylamide gel onto nitrocellulose or PVDF membrane.

  2. Incubate the membrane in a blocking buffer for 1 h at room temperature.

  3. Incubate the membrane with the antibodies diluted to the desired concentration for 2 h at room temperature or overnight at 4C.

  4. Remove antibody (they can be reused several times) and wash membrane 4 x 10 min in a blocking buffer.

  5. Incubate the membrane for 1 h in diluted secondary antibodies (use them only once).

  6. Wash the membrane 4 x 10 min in a washing buffer.

  7. Prepare the mixture of developing solutions 1 and 2 (1 : 1) and develop the membrane by incubation in that solution for 1 min.

  8. Expose the membrane with X-Ray film for a short time (1 min and longer).




Storing and testing antibodies

Storing non-purified antibodies

  1. Add 0.02% sodium azide to blood serum.

  2. Dispense the antibodies in convenient volumes and store at -20C. Most antibodies are stable for years when stored at -20C.

  3. Working antisera can be stored at 4C where they are stable for at least 6 months.

  4. Avoid often freezing and thawing of antibodies.




Analysis of obtained antibodies

Each obtained antibody requires careful characterisation. Perform at least two analyses in order to characterise your antibodies.

  1. Characterise the titre of antibodies required for your routine work. Dilute antibodies from 1 : 100 till 1 : 10.000 and perform Western analysis with each of these dilutions.

  2. Check whether the signal obtained on the Western blot is specific for your antigen or it is a non-specific signal. In that case you can block antisera with your antigen as described in the antigen competition assay.




ELISA procedure (antibody capture)

Enzyme-linked Immunosorbent Assay (ELISAs) provides a useful measurement of antigen or antibody concentration. The antibody capture assay performed in antigene excess can be used to determine the presence and level of antibodies. This method is specially recommended when polyclonal impure antibodies are used.

10 x PBS:0.75 M NaCl
30.0 mM KCl
45.0 mM Na2HPO4 x 12 H2O
15.0 mM KH2PO4
  
Blocking buffer:1 x PBS
5% low-fat dry milk (or optionally 3% BSA)
0.5% Tween 20
  
Washing buffer:1 x PBS
0.5% Tween 20


  1. For most applications, a polyvinylchloride (PVC) microliter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.

  2. Make 1 10 g/ml solution of antigen (peptide or protein) in 1 x PBS. PVC binds approximately 100 ng/well. If maximal binding is required, use at least 1 g/well (20 g/ml). Add 100 l of diluted antigen per well. Incubate the samples for 2 6 h at room temperature or at 4C overnight.

  3. Wash the plate twice with 1 x PBS.

  4. The remaining sites for protein binding on the PVC plate must be saturated by incubating with blocking buffer. Fill the wells to the top with blocking buffer. Incubate for 2 h to overnight in a humid atmosphere at room temperature.

  5. Wash the plate twice with 1 x PBS.

  6. Dilute preimmune (as a control) and immune sera (serial dilutions 1 : 100 to 1 : 100.000). Fill the wells with 50 l of the antibody test solution to antigen-coated wells. Incubate plates at room temperature for 2 h under gentle orbital shaking.

  7. Wash wells three times with 1 x PBS.

  8. Add diluted secondary antibodies. Follow recommendations of the antibody manufacturer. Incubate for 1 h at room temperature under gentle shaking.

  9. Remove the unbound secondary reagent by washing three times with 1 x PBS.

  10. Add the proper reagent available on the market. Follow the recommendations of the manufacturer.

  11. Read absorbance at appropriate wavelength (depends on the used substrate).




Antigen competition assay

  1. Dilute your antigen (peptide or protein) starting from 0.1 : 1 molar ratio between peptides and antibodies. In some cases you will need 100 fold excess of peptide to compete out the protein band on Western. Titration of peptide is important to find the right signal inhibition conditions.

  2. Add antibodies to the antigen and incubate for 2 h at room temperature or at 4C overnight.

  3. In order to reduce the background, centrifuge the solution for 15 min at 15.000 x g. Resulting supernatant can be used for further immunological analysis.

  4. Perform Western blot with the supernatant.

  5. If you get no signal on your Western blot with antibodies pre-incubated with the antigen or the signal will be stronger reduced it would mean that the signal is protein specific.


Note: If the conjugate peptide-carrier protein has been used to raise antibodies, using a conjugate in this assay might give false results.



Purification of antibodies

Purification on protein A columns (low salt)

This technique is applied for antibodies with high-affinity binding sites for protein A. Monoclonal antibodies from mouse IgG2a and IgG2b subclasses will bind well to protein A. Antibodies of the IgG3 subclass bind with intermediate affinity, but IgG1 molecules bind with low affinity. Most rat monoclonal antibodies cannot be purified using protein A columns.

Washing buffer 1:100 mM Tris-HCl pH 8.0
  
Washing buffer 2:10 mM Tris-HCl pH 8.0
  
Elution buffer:100 mM glycine pH 3.0
  
Collecting buffer:1 M Tris-HCl pH 8.0


  1. Adjust the pH of the crude antibody preparation to 8.0 by adding 1/10 volume of collecting buffer 1.

  2. Pass the antibody solution through a protein A bead column. These columns bind approximately 10 20 mg of antibody per ml of wet beads (one molecule of protein A binds two molecules of antibody). Serum contains approximately 10 mg/ml of total IgG.

  3. Wash the beads with 10 column volumes of washing buffer 1.

  4. Wash the beads with 10 column volumes of washing buffer 2.

  5. Elute the column with elution buffer. Add this buffer stepwise, approximately 500 l per sample. Collect the eluate in 1.5 ml tubes containing 50 l of collecting buffer. Mix each tube gently to bring the pH back to neutral. As with all protein solutions, avoid bubbling or frothing as this denatures the proteins.

  6. Identify the immunoglobulin-containing fractions by absorbance at 280 nm (1 OD = approximately 0.8 mg/ml).


Note: For analytical work in which more than one antibody will be purified on the same column, extreme care must be taken. Because different antibodies have such varied affinities for protein A, it is easy to contaminate one batch with the residual from a previous run. Washing columns sequentially with 2 M urea, 1 M LiCl, and 100 mM glycine (pH 2.5) will be sufficient for most needs.

Reference: Harlow E and Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory



Immunoaffinity binding of antibodies on an antigen column

Washing buffer 1:10 mM Tris-HCl pH 7.5
  
Washing buffer 2:100 mM glycine pH 2.5
  
Washing buffer 3:10 mM Tris-HCl pH 8.8
  
Washing buffer 4:100 mM triethylamine pH 11.5
  
Washing buffer 5:10 mM Tris-HCl pH 7.5
500 mM NaCl


  1. Covalently attach the antigen to a bead support. A number of different supports are available commercially.

  2. After coupling, carefully drain the resin and retain the supernatant. Measure the protein concentration to confirm that coupling has taken place. In most circumstances, efficient coupling will exceed 90%.

  3. Transfer the beads with the bound antigen to a column. Wash the column with 10 bed-volumes of buffer 1. Then wash with 10 bed-volumes of 1washing buffer 2, followed by 10 volumes of buffer 3. Check the pH of the last drops of the Tris wash. If it is not 8.8, continue the wash. Then add 10 bed-volumes of washing buffer 4 (prepared fresh). Wash with washing buffer 1 until the pH reaches 7.5.

  4. Pass the polyclonal serum through the column to bind the antibody. The serum should be free of any debris prior to loading on the column. If using the whole serum dilute the serum 1 : 10 in washing buffer 1 prior to loading. The antibody solution should be passed through the column three times to ensure complete binding, or should be applied at a slow flow rate if using a peristaltic pump.

  5. Wash the column with 20 bed-volume of washing buffer 1, and then with 20 bed volumes of washing buffer 5.


Reference: Harlow E and Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory



Elution of antibodies from antigen column

Elution buffer 1:100 mM glycine pH 2.5
  
Elution buffer 2:100 mM triethylamine pH 11.5
  
Collecting buffer:1 M Tris-HCl pH 8.0
  
Neutralisation buffer:1 M Tris-HCl pH 8.0
  
Washing buffer 1:10 mM Tris-HCl pH 8.8
  
Washing buffer 2:10 mM Tris-HCl pH 7.5


  1. Elute the antibodies that are bound by acid-sensitive interactions by passing 10 bed-volumes of elution buffer 1 through the column. Collect the eluate in a tube containing 1 bed-volume of collecting buffer.

  2. Wash the column with washing buffer 1 until the pH rises to 8.8.

  3. Elute the antibodies that are bound by base-sensitive interaction by passing 10 bed-volumes of elution buffer 2 (prepared fresh) through the column. Collect the eluate in a tube containing 1 bed-volume of collecting buffer.

  4. Wash the column with washing buffer 2, until the pH is 7.5. The column may be reused by storing it in buffers with 0.01% merthiolate.

  5. Combine the antibody fractions and dialyze against PBS with 0.02% sodium azide. If necessary, concentrate the antibody solution by ammonium precipitation or by running a protein A column.


Reference: Harlow E and Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory



Antibody purification from immunoblots (small amounts)

10 x PBS:0.75 M NaCl
30.0 mM KCl
45.0 mM Na2HPO4 x 12 H2O
15.0 mM KH2PO4


  1. Run preparative SDS-PAGE with loaded antigen.

  2. Transfer antigen protein(s) onto nitrocellulose or PVDF membrane.

  3. Block the membrane, incubate it with the primary antibodies and wash with washing buffer (according to the standard Western blot procedure).

  4. Cut the strip of the blot from the side and perform normal antigen detection.

  5. Store the main body of the blot in 1 x PBS at 4C.

  6. After the localisation of the antigen-primary antibody complexes on the side strips, align the strips with the untreated portion of the blot.

  7. Excise the area of the blot that contains the antigen and primary antibody using a sharp scalpel.

  8. Cut this into small pieces and transfer to a test tube. Incubate with 100 mM glycine (pH 2.5) for 10 min or with 1 M KSCN for 10 min.

  9. Remove the buffer and neutralize with one-tenth volume of 1 M Tris-HCl (pH 8.0) for the glycine elution or dilute with 10 volumes of 1 x PBS for the KSCN elution. The antibodies are now ready for further tests.


Reference: Harlow E and Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory






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