Version: 5/12/09

WESTERN BLOTTING WITH ECL DETECTION

READ THE WHOLE DOCUMENT BEFORE ATTEMPTING THIS PROCEDURE!

There are two western blot procedures you can try, one uses NETG buffer and the other TTBS buffer. We have found that the NETG procedure gives somewhat lower background than TTBS so it is recommended that you first try the NETG procedure. Information about preparing a cell lysate is given at the end along with other key information about optimizing your western blot.

NETG Procedure: Run SDS-PAGE Laemmli minigel (using 0.75 mm gel spacers) with the % acrylamide that is appropriate for the separation of your proteins of interest (i.e. 14% gel is good for sPLA2s, MW ~14-18 kDa). Run the gel at 200 Volts (constant). It is a good idea to always use 1 gel lane for prestained MW markers. After running gel, rinse gel in water for 1-2 min in a plastic tray (a convenient tray to use is the plastic covers that fit over racks of Pipetman tips) then transfer gel to tray of electrotransfer buffer (per liter 3.03 g Tris base (don't use the HCl salt), 14.4 g glycine, 20% methanol (v/v) with or without SDS (see below), note %SDS calculated based on total buffer volume, i.e. including methanol, pH does not need to be adjusted, it will read ~8.3 if you check it). Allow gel to soak in transfer buffer for 15 min. (note longer soak times are needed for gels thicker than 0.75 mm, see BioRad electrotransfer manual). Note, if desired, you can run 2 minigels at the same time and also electrotransfer 2 gels at the same time.

To prepare PVDF membrane (PolyScreen PVDF, NEN, Cat. # NEF1000, handle with gloves), fill a plastic tray with methanol and with tweezers lower the membrane into the methanol at 45 deg angle until it is completely submerged, after 15 sec grab the membrane with tweezers, poor off the methanol and fill tray with milli-Q water, swirl briefly and pour off water, then soak in electrotransfer buffer for 10 min, then place in BioRad Mini Protean II electrotransfer unit against gel (read BioRad electrotransfer unit manual). (NOTE: we have had problems with new batches of the fiber pads (from BioRad) that are used in the electrotransfer unit (received in late 2002) in that the pads are much thicker than usual, especially in the middle of the pad, and the electrotransfer does not work with these pads. We are trying to get a solution to this problem from BioRad.) Electrotransfer for 60 min at 100 Volts (constant) with cooling with ice. Note, prechill the transfer buffer before filling the electransfer tank, place the gel-blot sandwhich in the holder into the tank, and place the entire assembled device in a styrofoam box full of ice, the ice should be all the way up to the lid of the transfer tank.

After electransfer, separate membrane from gel (forceps and gloves) and hang the membrane on a string using a clothespin and allow membrane to air dry at room temp. for 30 min. Once the blot is dry you can continue with the procedure or store it by laying the membrane on a piece of Whatmann 3MM paper, wrapping with Saran Wrap and leaving in the fridge for several months if not longer.

To process the blot (all steps done in plastic tray at room temperature with gentle shaking on an orbital shaker and with side of membrane containing transfered proteins facing upward), re-wet for ~15 sec with pure methanol, pour off methanol, rinse briefly with Milli-Q water, pour off water and add a known volume of NETG buffer (see below for recipe) (enough to suspend the blot well enough so that it moves freely during shaking, typically 20 ml). Allow blocking to proceed for 2 hr, then pour off the buffer, add known volume of fresh NETG buffer (typically 20 ml) and add primary antibody to desired final concentration directly to the NETG buffer above the blot (this is why you need to know the volume of NETG buffer added), continue shaking 1 hr, wash using the following schedule (note details are important to get a good blot with low background): drain off buffer by tipping the tray and allowing the wet blot to adhere to the tray bottom, hold long enough til buffer stops dripping out, no need to shake. Add back 30 ml (i.e. 50% more volume than used previously) of NETG buffer and swirl for 6 sec (by hand in this case), drain off buffer and fill with 30 ml NETG buffer and swirly by hand for 20 seconds, drain off buffer, add back 30 ml NETG buffer and swirl on orbital shaker for 1 min, drain, add back 30 ml NETG, swirl 3 min, drain, add back 30 ml NETG, swirl 10 min, drain, add back 30 ml NETG buffer, swirle 30 min. Add back known volume of NETG buffer (typically 15 ml) and add secondary antibody (see below for source of secondary antibody) to desired final concentration, swirl 1 hr, and wash with NETG buffer exactly as above.

Take the blot in the tray containing the last wash solution to the dark room or near the dark room and process the blot for ECL detection. Mix equal volumes of the two ECL reagents (Amersham) in a 15 ml Falcon tube (cap tube and invert a few times) (note the reagents are stored in the fridge and allowed to warm to room temp. before use). You need 2 ml (i.e. 1 ml of each ECL reagent) for a single membrane from a single mini-gel. With forceps, pull the blot out of the tray with the last wash solution and let it drain briefly by touching its edge to a KimWipe.

DO NOT LAY THE BLOT ON THE KIMWIPE, THIS WILL LEAVE DUST PARTICLES ON THE BLOT WHICH WILL GIVE A VERY SPOTTY BACKGROUND

After draining the membrane on the Kim Wipe, place membrane protein side up onto a piece of Saran Wrap laying on the lab bench (no wrinkles and the bench should be level), add the mixed ECL reagents (2 ml per membrane from a mini-gel) with a P1000 pipettor so that the liquid is held by surface tension over the entire membrane surface. Leave the membrane for exactly 1 min at room temp. without agitation. Using forceps (with gloves) drain off excess ECL reagent by holding the edge of the membrane against a KimWipe (again don't lay blot on KimWipe) for a few seconds to drain off reagent. With forceps, lay the membrane protein side down on a new piece of Saran Wrap (wrinkle free, have it ready before applying ECL reagents) and close the Saran Wrap all the way around the backside of the membrane to completely seal the membrane between two layers of the Saran Wrap. Trim off excess Saran Wrap with scissors, place wrapped membrane protein side up into film cassette, turn off room light, turn on safety light and lay a piece of x-ray film over the Saran Wrap covered blot (it is critical not to get the film moist with ECL reagents so avoid leakage of reagents out of the wrapped membrane). Use the Hyperfilm ECL (Amersham, stored 4 deg C, check expiration date). Close cassette and expose for 20 sec, open cassette and exchange film for a new film and expose second film typically 1 min, develop both films. Examine both films, if you want more sensitive detection (if the background allows this) you can try a third film exposure for 3 min and even a 4th film exposure for 10 min.

TTBS Procedure: See NETG procedure for the basic steps of western blot processing. After electrotransfer (see NETG procedure), rinse blot twice (1 min each) with milli-Q water. Transfer the blot to a tray of TTBS (recipe given below) with 5% milk (BioRad Milk, Blotting Grade Blocker Non-fat Dry Milk, Cat. 170-6404) and block either 2 hr at room temperature or overnight at 4 deg in cold room with gentle shaking. Rinse blot two times with TTBS (~ 30 sec each) at room temperature, add back known volume of TTBS and add antiserum to desired final concentration, incubate for 2 hr at room temp., wash as above (see NETG procedure) with TTBS, add back known volume of TTBS and add secondary antibody to desired final concentration, incubate 1 hr at room temp, wash as above (see NETG procedure) with TTBS and prepare for ECL as described above for NETG procedure.

Reagents:

NETG buffer: 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4, 0.05% Triton X-100, 0.25% Gelatin (BioRad Cat. # 170-6537). Warm slightly to dissolve gelatin, filter it though a 0.45 micron Nylonn-66 solvent filter (use FPLC solvent filter glass device, be sure to rinse filter device well after use to remove gelatin and detergent), Make NETG buffer fresh each 24 hr period, during the day OK to store at room temperature, if you want to use it the next day, store in the fridge overnight.

TTBS buffer: Make 10X TBS buffer (per liter: 80 g NaCl, 2 g KCl, 30 g Tris base (not HCl salt), adjust to pH 7.4). Store 10X TBS at 4 deg C. To make TTBS, dilute 10X TBS 10-fold with water and add 0.75 ml Tween-20 per liter, make TTBS as needed, OK to store at 4 deg C or room temp. for a few days.

Note, at times we have used our own homemade ECL reagents rather than the Amersham reagents. Amersham reagents are fine, if we want to do a very large number of blots and save some money, here is the recipe for making the ECL reagents.

Homemade ECL reagents:

30% hydrogen peroxide (Reagent Grade, store 4 °C)

p-coumaric acid (ICN Cat. # 102576)

luminol (Sigma A8511)

Stock solution of luminol (0.11 g/ml DMSO), store 4 °C for up to 1 week (tube wrapped with foil).

Stock solution of p-coumaric acid (0.09 g/ml DMSO), store 4 °C for up to 1 week (tube wrapped with foil).

Prepare Homemade ECL mixture as follows:

Make fresh: In a 50 ml Falcon tube, add 25 ml 0.1 M Tris, pH 8.6, add 20 ul 30% reagent-grade hydrogen peroxide, mix well. Wrap a second 50 ml Falcon tube with foil, add 25 ml 0.1 M Tris, pH 8.6, add 60 ul of p-coumaric acid stock solution (see recipe below), mix well, add 100 ul of luminol stock solution (see recipe below), mix. Immediately before use, mix the contents of both Falcon tubes together. Be sure both solutions are at room temperature before mixing them together. Pour the 50 ml into a new plastic tray. Note this tray of 50 ml can be used to process up to 2 blots, if you have more blots, make a new portion of ECL mixture.

Secondary Antibody:

The anti-rabbit IgG and anti-mouse IgG ECL II° Abs are from Amersham-Pharmacia Biotech. We use it typically at a dilution of 1/3,300.

OPTIMIZATION OF ELECTROTRANSFER AND POSTIVE CONTROL WITH ADDED RECOMBINANT ANTIGEN.

With western blotting, it is fairly common that you easily detect your antigen when you load it alone on the lane of a gel but you fail to see it when you spike your sample to be analyzed (i.e. cell lysate) with the antigen. For this reason, you MUST always perform the positive control of not only runing a lane of pure antigen but also of sample to be analyzed, i.e. cell lysate, spiked with the same amount of antigen.

Another reason that you don't see the antigen in the spiked lysate is because of problems during electrotransfer. If your antigen transfers quickly, other proteins may transfer later and cover your antigen on the blot so it cannot bind to antibody. You can try electrotransfer for 20 or 40 min instead of the usual 60 min. If that does not work, you can try adding 0.01% or 0.1% SDS to the transfer buffer. Electrotransfer optimization may be required for each individual antigen you are analyzing. So considerable work is sometimes required to get reliable western blots to work. Thus you need to optimize this by using recombinant antigen together with cell lysate before you attempt to detect endogenous antigen in the cell lysate. Typically 1-2 ng of recombinant antigen is used for spiking (more if the primary antibody is not able to detect down to the 1-2 ng level). Thus before doing cell lysis spiking, it is important that you know the range of antigen amount that your antibody can detect in a blot done with recombinant antigen alone on the gel lane (no cell lysis). By running a gel of recombinant antigen alone, you will know exactly where on the gel to look for the western blot band due to the spike added to the cell lysate. This is important because for most western blots, other protein bands will be detected, they are almost never perfectly clean, especially when a cell lysate is added.

Note: to add 1-2 ng of spike protein to a sample, you will have to prepare a highly diluted stock solution of recombinant protein. Your protein may stick to the walls of the tube when stored as a highly dilute solution (i.e. typically << 0.1 mg/ml). See tips below for making dilute protein stock solutions in Laemmli sample buffer.

MAKING A CELL LYSATE AND THE AMOUNT OF PROTEIN TO LOAD PER GEL LANE

Here is the procedure we have used for making cell lysates from human lung macrophages. This is a good general purpose procedure for making lysates from cells for western blotting.

1. Spin down the cells and aspirate off the culture medium.

2. Resuspend cell pellet in balanced salt solution (i.e. PBS or whatever BSS is prefered for your cells), spin down the cells again and aspirate off the supernatant.

3. Resuspend the cells in balanced salt solution and distribute into separate tubes, typically 1 million cells per tube. Spin down the cells and aspirate off the supernatant.

4. To each cell pellet add 20 ul of ICE-COLD buffer A (66 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 25 mM NaF, 1 mM PMSF, 10 ug/ml leupeptin, 10 ug/ml aprotinin). Freeze suspension in dry ice-ethanol bath and store tubes at -80 deg C or ship on dry ice to receiving lab (obtain receipt of the package, transfer tubes from dry ice to -80 freezer without letting tubes thaw). Note that buffer A should be stored frozen since it has protease inhibitors.

5. We find it is problematic to sonicate the tubes of 1 million cells (see above) because of losses due to foaming and it is hard to fit the microtip sonicator probe into such as small volume. So we allow all tubes (or a subset of tubes depending on the number of cells needed) from the same batch of cells to thaw on ice, resuspend the cell suspensions by pipetting up and down and then combine all cell suspensions into 1 eppendorf tube.

6. To this single tube of combined cells, on ice, measure volume with the pipettor and add appropriate volume of ice-cold 4X Laemmli loading buffer (without dye or thiol).While keeping tube on ice, sonicate the sample with the microtip for 6 x 15 second bursts, with 45 seconds off time in between each burst (the sonicator should be tuned according to the instruction manual, this involves first tuning the instrument with the microtip removed and replaced with the screw-in cap that attaches to the horn, once instrument is tuned, just replace screw-in cap with microtip, make sure that the contact surfaces are clean so that they sit well against each other).

7. Microfuge the tube at full speed in the microfuge in the cold room for 15 min. Transfer all of the sup. to a new eppendorf tube and spin this full speed for 15 min in the cold room. Transfer all of the sup. to a new eppendorf tube, always on ice.

8. Use a small aliquot of sup. (typically 1 ul) to measure the protein concentration (BioRad Bradford dye binding assay with BSA as a standard). The typical protein concentration is about 8 ug/ul. This can be aliquoted into eppendorf tubes and stored at -80 deg C until analyzed by western blotting.

9. Prior to loading sample onto the gel, take the appropriate volume of cell lysate and add the appropriate amount of sPLA2 spike if desired.We typically load 10-20 ug of total cell protein per lane of a mini-gel. If the sample is to be processed under reducing conditions (see below), freshly add neat beta-mercaptoethanl to give 5% by volume, mix by flicking the tube a few times. If sample is to be boiled (see below), place in boiling water bath for 4 min (you can use the special eppendorf tubes that have the lock-down lids).

10. Load sample onto the gel lane, we load 10-20 ug of total cell protein per lane, typically in 20 ul. It is important to record how much protein was loaded per gel lane and if you want to compare the western blot band intensity between two different gel lanes, it is important to load the same amount of total cell protein on each lane and to put both samples onto the same gel (it is not reliable to compare western blot band intensities for samples run on different gels and thus on different blot membranes).

THIOL OR NOT IN THE LAEMMLI SAMPLE BUFFER AND BOILING OR NOT

The ability of your antisera to detect your protein may depend on whether your protein is reduced (thiol treated prior to loading onto the Laemmli gel) or not. Often samples with or without thiol are boiled prior to loading onto the gel. We have found for some antigens that the protein of interest is not seen when the cell lysate sample containing the protein spike is boiled. Perhaps the protein of interest aggregates with other proteins in the lysate during boiling. Thus, you need to determine in advance whether your antibody detects the reduced versus non-reduced protein and whether the spike is detected after boiling or not. These parameters are very important and this serves as another reminder that a positive control with the SPIKED cell lysate is critical for reliable interpretation of the western blot.

HOW MUCH PROTEIN PER GEL LANE

You may be able to push the limit of detection of your protein by western blot by loading more than 10-20 ug of cell protein per mini-gel lane. However, we find that loading much more than 20 ug of protein per lane leads to loss of resolution in the gel (commassie staining reveals some smearing of the protein bands). Also we have found that for many antigen-antiserum pairs, the western blot signal detected for say 1 ng of antigen loaded alone onto the gel is much stronger than the signal obtained from 1 ng run in the same gel lane with the cell lysate protein (as discussed above). Thus adding more than 10-20 ug of protein per mini-gel lane will probably not help to increase the western blot detection sensitivity. You can try a few different gel lanes, say 10 ug of cell protein in lane 1, 20 ug in lane 2, 30 ug in lane 3, 50 ug in lane 4, all spiked with 1 ng of antigen, then see if the detection of the 1 ng is constant for all lanes or whether the signal gets weaker in the lanes with higher cell protein. Based on this, you can decide if loading up to 50 ug of cell protein per lane will be beneficial in terms of pushing the limit of detection as high as possible.

For additional tips on Western blotting you can look at the handbook from Millipore. Millipore Protein Blotting Handbook

WESTERN BLOTTING AFTER IMMUNOPRECIPITATION

If you immunoprecipitate your antigen from a biological sample in order to enrich the sample to load onto the gel for a western blot, it is important to remember that you will be loading quite a lot of anti-antigen IgG onto the gel for the western blot. The heavy and light chain of the IgG will be transferred to the blot and will be detected as strong interferring bands on the western blot film since the HRP-anti-IgG secondary antibody will bind to either the heavy chain, the light chain or both depending on what kind of secondary antibody it is. The light chain band is around 25 kDa and the heavy chain band is around 55 kDa. You may also see strong bands on the film between 25 and 55 kDa, these could be protein A or protein G that leaches off the beads used for the immunoprecipitation, these proteins may also bind your secondary antibody and give strong background bands. If faster than 25 kDa or slower than 55 kDa you may be OK but if you antigen is near the 25-55 kDa region you will likely not see the band because of the strong interferring bands. We have found that the kit from Genscript for immunoprecipitation/western blot works well. Order the kit that is appropriate for the species in which your primary antibody is made in, for example the rabbit IgG kit is Cat. number L00231. The kit is sufficient for about 10 blots. But the blotting paper that comes with the kit (8 x 7.5 cm) is too big for our minigels so we cut the blot paper to the appropriate size (8 x 5.2 cm). We follow the kit instructions but reduce the amount of reagents used by appropriate factor based on the area of your blot membrane compared to the area of the membrane supplied in the kit. Also the kit says to place the membrane on a soft tissue, instead we hold the blot vertically with its edge against a Kim-Wipe to let the excess liquid drain off the blot. Note also that if the immunoprecipitation is done with protein A/G beads you need to use the protein A/G blocking reagent that comes with the kit.