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Day 6: Isoelectric Focusing and Western Blot Development

Assignments Due


Overview of Experiment

Isoelectric focusing (IEF) is used to determine the isoelectric point of ADA: the band corresponding to adenosine deaminase is located by a colorimetric activity stain specific for the enzyme.  The western blot is developed by using antibodies that have been raised in goats against the native mouse adenosine deaminase: these antibodies bind to protein bands that correspond to adenosine deaminase and are located by using a conjugate antibody system consisting of anti-goat IgG antibodies covalently linked to alkaline phosphatase; the phosphatase reacts with BCIP producing a purple product.  Molecular mass (m) of both native and recombinant ADA is estimated from a standard curve.


Isoelectric Focusing

Attaching the gel support film to the glass plate

  1. Place the clean glass plate on a paper towel. The towel serves to soak up the water squeezed from beneath the membrane and provides a slightly padded surface.
  2. Place a narrow line of water across the middle of the plate.

  3. The gel support film has two surfaces, a treated hydrophilic side which the acrylamide adheres to, and a hydrophobic side.
    The film will be given to you with the hydrophilic side up; if you forget which side is which, put a drop of water on the edge of the film: it beads on the hydrophobic side and spreads on the hydrophilic side.

    Bend the support film U-shaped and place hydrophobic side DOWN against the glass plate. Relax the bend and align the membrane with the edges of the plate. The membrane must not extend past the edges of the glass.
  4. Roll with a clean test tube with significant downward pressure to squeeze out as much water as possible. Avoid wetting the hydrophilic side of the membrane.
  5. Carefully blot off any excess liquid at the edges.

Preparation of the polyacrylamide gel

***WARNING: unpolymerized acrylamide is a potent neurotoxin! always wear gloves and dispose of solution carefully.***
  1. Prepare the monomer-ampholyte solution in a 125 ml sidearm flask.
    *Flasks, 1 per two groups, with rubber stoppers are available on the reagent bench.
  2. Degas the solution for 5 minutes using the vacuum in the hood; do not degas longer since some O2 is required to catalyze riboflavin-5'-phosphate (FMN).
    *Disconnect the flask from the vacuum source BEFORE turning off the vacuum.
  3. Add the catalyst solutions and swirl gently.
  4. Pipet the gel solution between the glass plate and the casting tray: see Casting the gel below.
  5. Fill the disposable transfer pipet with any remaining acrylamide solution and discard in the trash; rinse the flask with RO water and return the flask and stopper to the reagent bench.

The following volumes are for casting TWO 125x65x0.4 mm gels (cast ONE gel per team):

Monomer-ampholyte solution


5.5 ml

acrylamide monomer concentrate
(25% T, 3% C)

2.0 ml

25% (w/v) glycerol

2.0 ml

amphloyte (pH 3/10 or pH 4/6, Bio-Rad)

0.50 ml

Catalyst solutions

10% (w/v) ammonium persulfate

15 µl

0.1% (w/v) riboflavin 5'-monophosphate (FMN)

50 µl

TEMED (neat)

3 µl

Casting the gel

  1. Put the glass plate GEL SUPPORT FILM SIDE DOWN in the casting tray.
  2. Fill a disposable plastic transfer pipet with the acrylamide solution.
  3. Generate and maintain a puddle of liquid on the casting tray just off the corner of the glass plate. It is not necessary to sweep or move the pipet tip during dispensing. The liquid will be drawn between the membrane and casting tray by capillary action.

    If the casting tray is very clean there should be no problems with air bubbles forming. Release solution slowly to prevent bubbles; if a bubble forms, carefully move the plate sideways until the bubble is at the edge.

  4. Set the tray in sunlight for 45-60 minutes.
  5. To remove the gel, lift one corner with a flat spatula inserted between the gel and the casting tray. Air will appear under the gel and then you can gently lift the glass plate from the casting tray. Do not apply too much pressure or you'll chip the corner of the glass plate.
  6. Flip the plate over so gel side is facing up.
***NOTE: It is important not to let the gel set out for more than 20 minutes before starting the run. The gel can dry resulting in insufficient water to carry the current for proper electrophoresis. Plan your work in advance - be prepared to load the samples and get them running without delay.***

Loading samples

  1. A sample template will not be used. Just apply your samples onto the surface of the gel as droplets (2 µl of standards, 4 µl of samples) leaving sufficient space between each to prevent the drops from merging. There is no need to wait for the samples to soak in.

  2. Remove the graphite electrodes from the electrophoresis cell and rinse them with RO-H2O; place the electrodes back into the cell and lightly moisten them with RO-H2O.
  3. Place the gel, SAMPLES SIDE DOWN, directly on top of the electrodes.
    Do not remove the glass plate from the gel support; the weight of the plate helps maintain good contact between the gel and the electrodes.
  4. Carefully slide the lid onto the IEF cell and plug the cables into the power supply.

Running the gel

*Isoelectric focusing is carried out under constant voltage conditions in a stepped fashion: **Up to three different sets of running conditions can be programmed and run in sequence with the Bio-Rad Model 1000/500 Constant Voltage Power Supply; the power supply automatically switches to the next step and turns itself off at the end of the last step.
  1. Turn on the power supply; switch is located on the right side.
  2. Press PROGRAM until S1 is lit.
  3. Set the voltage and the time (in hours).
  4. Press PROGRAM once so S2 is lit.
  5. Set the voltage and the time (in hours).
  6. Press PROGRAM once so S3 is lit.
  7. Set the voltage and the time (in hours).
  8. Press PROGRAM until S1 is lit.
  9. Press the RIGHT SELECT button until DISPLAY is lit to monitor the actual voltage (v) and current (ma) during the run. As the focusing nears completion, the current decreases to near zero.
  10. Press RUN.

***IMMEDIATELY after the run is completed, cut the gel in 1/2 using scissors and proceed with activity stain.***
Keep gel-side UP; if you put gel-side DOWN, the gel will stick to any surface and will be ruined.

BIO-RAD IEF Standards





Phycocyanin (3 bands)






Beta-lactoglobulin B




Bovine carbonic anhydrase




Human carbonic anhydrase




Equine myoglobin (2 bands)





Human hemoglobin A




Human hemoglobin C




Lentil lectin (3 bands)






Cytochrome C




NOTE: These standards must be recorded in your lab notebook.

Activity Stain of IEF Gel

This colorimetric activity assay uses an enzyme-coupled system and is specific for ADA.

Stock Solutions

100 mM Tris-HCl, pH 8.0, 10 mM Na arsenate, containing 10 mM adenosine
xanthine oxidase (0.01 U/µl) and purine nucleoside phosphorylase (0.08 U/µl) = (XO:PNP) [*enzymes purchased from Sigma-Aldrich]
10 mg/ml nitroblue tetrazolium chloride (NBT)
1 mg/ml phenazine methosulfate (PMS)
1% agarose solution (melted then held at 50°C in a water bath)


Read over this section carefully and be prepared to complete the protocol quickly; solution volumes are for a 1/2 gel.

  1. With 10-15 minutes remaining on the IEF run, incubate 2.75 ml of Tris/arsenate/adenosine solution at 37°C in a 100 ml beaker.
  2. As soon as the gel electrophoresis is finished, complete the detection solution by adding the following to the incubated Tris-substrate solution:
    10 µl XO:PNP


    300 µl NBT solution and 300 µl PMS solution
    NOTE: do NOT add NBT and PMS until you are ready to pour the agarose overlay (i.e., add the agarose and proceed with step 3.); these compounds are light sensitive and will turn "purple" if exposed to light for too long

    Mix well

    NOTE: Be frugal with the solution because the enzymes used in this assay are moderately expensive.

    Slowly add 2.7 ml of melted 1% agarose solution from the 50°C bath; use a disposable transfer pipet with the bulb squeezed completely in to deliver ~ 2.7 ml.
    Let the agarose flow down the side of the 100 ml beaker to aid in cooling to prevent denaturation of the enzymes. Swirl gently to mix but avoid introducing air bubbles into the solution.

    Be prepared to complete step three immediately; the solution may solidify within 5-10 seconds.
  3. Immediately, pour the molten detection mixture evenly over the gel that you wish to stain for activity. Just pour from the beaker and "puddle" the reaction solution over the surface of the gel. Place the gel on a plastic lid while applying the gel solution and allow the slurry to harden before moving the gel. Keep the slurry on the gel. Any material off the gel is wasted. Air bubbles may be coaxed to the edge or popped using the bulb of a transfer pipette.
  4. Incubate the gel in the dark (PMS and NBT are extremely light sensitive; protect these solutions from light as much as possible) at 37°C.
  5. Check the development after 20 minutes: if specific bands are present, photograph the gel for your records (each team should bring a DIGITAL CAMERA to lab--iPhone/iPad will NOT work); if no distinct bands are visible, let the reaction proceed for an additional 10-15 minutes--after this time, if only nonspecific staining is present, discard the gel (no picture is necessary).  
  6. Estimate the pI from the relative position of the ADA band compared to the standards.

Develop the immunoblot (western blot)


Tris buffered saline (TBS): 10 mM Tris-HCl, pH 8.0, 150 mm NaCl
TBS + 0.05% Tween 20 (TBST)
Blocking solution: 1% (w/v) Carnation instant milk solution in TBST
Staining solution: premixed alkaline phosphatase conjugate substrate solution (Bio-Rad) contains nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP) in buffer; protect solution from light.


Small volumes (~ 10 ml) are used for preparation of the membrane. Place the membrane in the small plastic box to ensure complete coverage of the membrane surface.

  1. Wet the PVDF membrane in methanol for 1-2 seconds. Rinse briefly with water and immediately place in the blocking solution. If the membrane "dries" and becomes opaque white or blotchy, wet with methanol again. The white area will not wet in aqueous solutions during the incubations.
  2. Block excess protein binding sites by placing the membrane into 10 ml blocking solution for at least 1 hour on the rocking shaker.
  3. Pour off the blocking solution and rinse blot with TBST (to remove residual milk).
  4. Bind the primary antibody (goat anti-mouse ADA antibody, from Bethyl Laboratories, Inc., Montgomery, TX) diluted 1:10,000 in TBST: a 10 ml aliquot of diluted antibody will be provided; incubate for 30 minutes on the rocking shaker.
  5. Wash for 3-5 minutes in TBST. Repeat two times.
  6. Bind the secondary antibody (anti-goat IgG-alkaline phosphatase antibody produced in rabbit, from Sigma-Aldrich) diluted 1:10,000 in TBST: a 10 ml aliquot of diluted antibody will be provided; incubate for 30 minutes on the rocking shaker.
  7. Wash the membrane as in step 5.
    (Optional step (we do not use): To decrease nonspecific interaction of the antibodies to some proteins, salt (NaCl) may be added to the wash buffer as high as 0.6 M concentration.)
  8. Blot membrane on paper towel to remove excess liquid.
  9. Transfer membrane to staining solution and incubate for about 5 minutes. Monitor the development -- this is not an end point detection system.
  10. As soon as you see purple bands first appear, rinse with RO water for a few minutes to stop the reaction.
  11. Let blot dry at room temperature; take blot with you (either scan or make enlarged copy of membrane for analysis).


Proteins are denatured by boiling in the presence of excess SDS and beta-mercaptoethanol; since SDS-treated proteins have essentially identical charge:mass ratios, these denatured proteins separate strictly according to size through polyacrylamide gels.  Under these conditions, a plot of log(10) molecular mass vs. relative mobility (Rf) shows a linear relationship (1, 2).
NOTE: for any given gel concentration, the relationship between log(10) molecular mass and mobility is linear over only a limited range (for a 10% gel the range is 15,000-70,000 Da).

Since the molecular mass standards are electrophoresed on the SAME gel as the unknown proteins, a STANDARD CURVE can be constructed by plotting log(10) molecular mass of the standard (Daltons) vs. distance migrated by the standard (measured from the TOP of the gel). (You do NOT need to determine Rf's unless your dye front is distorted.)
NOTE: this graph must be hand-drawn in your laboratory notebook. although calculators/computers can generate the curve for you, by plotting the curve manually you can ensure that your unknown lies on the linear (and therefore valid) portion.

***You have to construct a standard curve to determine the molecular mass of recombinant and native ADA.***

NOTE: Molecular mass (symbol "m") is expressed in daltons (Da); one dalton is 1/12 of the mass of carbon 12. Apparent molecular weight (Mr, relative molecular mass) is the ratio of the mass of a molecule to 1/12 of the mass of carbon 12 and is dimensionless; hence, it is not correct to express Mr in daltons. Report your results for recombinant and native ADA as molecular mass (m), in daltons or kilodaltons.

  1. Shapiro, A.L., Vinuela, E., and Maizel, J.V. (1967). Molecular estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun.,28: 815-820.
  2. Weber, K. and Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem.,244: 4406-4412.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 16 June 1999
Updated 12 September 2016