Western Blotting
ELECTROPHORESIS
Prepare and run an SDS PAGE gel. Select a gel percent which will give the best resolution for the size of antigen being analyzed (if known). If the size is not known, a 12% gel is a good starting point. Load enough protein to provide 0.1-1ng of antigen per well. It is generally advantageous to load serial dilutions of sample, so as to ensure that one lane at least will fall in the optimal range for detection. If desired, use prestained markers, such as National Diagnostics' ProtoMarkers, to monitor transfer efficiency.
TRANSFER
Upon completion of the run, the gel must be transferred onto a blotting membrane. This can be accomplished by semi-dry transfer or wet transfer. Both are electrophoretic transfers, and require equipment designed for that purpose. The procedures outlined below are intended as general outlines. For best results and to ensure safety, follow the equipment manufacturer's instructions for this phase.
SEMI-DRY BLOTTING
1. Rinse electrode plates with deionized H2O.
2. Cut 6 sheets of Whatman 3MM paper and 1 sheet of blotting membrane to the size of the gel, or slightly smaller.
3. Wet the membrane. Soak nitrocellulose in deionized H2O for 3 minutes. Soak PVDF in methanol for 1 minute. Transfer membrane to transfer buffer and soak for 3 minutes.
Transfer buffer: / 0.02M Tris base0.15M Glycine
20% MeOH
ASSEMBLE THE TRANSFER STACK
1. On the lower plate (positive, red lead), place in order:
i. 3 sheets of 3MM (precut and wetted with transfer buffer)
ii. Transfer membrane
iii. Gel
iv. 3 sheets 3MM (precut and wetted with transfer buffer)
Remove any air bubbles by rolling over each layer as placed with a 10ml pipette. Be careful not to disturb the stack, or to let the gel stick to the pipette. Roll gel after placing first upper layer of paper, if desired. Note that bubbles trapped in the stack will distort current flow, leading to lateral band displacements and failure of bands to transfer.
2. Check to be sure that no portion of the upper paper stack contacts the paper or the electrode underneath the gel. Contacts between upper and lower stacks will short circuit the current, distorting the transfer. In some systems, parafilm or plastic wrap may be arranged around the gel to prevent this short circuiting from occurring. Check the instructions.
3. Place the upper (negative, black lead) electrode plate on top of the stack, and apply current. Consult apparatus instructions; typical conditions are 1 hour @ 0.8 mA/cm2. Over-transfer may dry the gel and drive proteins through a Nitrocellulose membrane.
WET TRANSFER
1. Cut 2 sheets of Whatman 3MM paper and 1 sheet of transfer membrane to the size of the gel.
2. Wet the membrane: Soak Nitrocellulose in water for 2 minutes. Soak PVDF in methanol for 2 minutes.
3. Place membrane in Transfer buffer (see "Semi-Dry Blotting" above)
4. Assemble transfer "sandwich" in order:
i. Filter paper sheet
ii. Gel
iii. Membrane
iv. Filter paper sheet
5. Assemble sandwich clamps and support pads as per equipment manufacturer's instructions.
6. Place sandwich in transfer tank with membrane side closest to the positive electrode (Red lead).
7. Add cold Transfer buffer, and initiate cooling procedure.
8. Apply voltage: This parameter is entirely dependent upon the apparatus used. Large format gels may require 50 - 75V, 5 - 15 hours. Minigels can be blotted in 1 hour @ 50 - 100V in some systems.
POST TRANSFER
It is prudent to mark the blot in a permanent way for orientation, by notching or clipping a corner. If prestained markers were used, their positions should be marked in pencil or an alcohol indelible ink. Often well positions can be distinguished and marked immediately after transfer.
Notes: Nitrocellulose membranes may be air dried prior to further processing. It has been reported that this improves protein retention on the blot. After transfer, Coomassie staining of the gel can give information about the efficiency of transfer.
1. Stain the blot and mark the positions of well and markers. If prestained markers were used, this step may be skipped.
2. At this point it is very helpful to spot diluted primary and secondary antibody on an unused area on the blot. This can be invaluable for troubleshooting. If a blot fails (no bands are detected) the antibody spots can be interpreted as follows:
VISIBLE / INTERPRETATION1° & 2° / 2° antibody functioning well - label okay, 1° antibody may have failed.
2° only / 2° antibody failed to bind 1°
no spots / Label enzyme denatured - remake 2° antibody dilution.
BLOCKING
A variety of blocking reagents are available. It is worthwhile to optimize blocking procedures, as this step determines the background level of the blot, and hence the detection limit. The most universal blocking agents contain mixtures of proteins and surfactants. This combination provides good to excellent blocking on most membranes.
1. Blocking Solutions:
1. ProtoBlock: Dissolve 10g of Reagent B in 170 ml deionized water. Add 20 ml of Reagent A.
2. Tween/milk: Dissolve 50g nonfat dry milk and 2 g Tween 20 in 1L PBS. If product is to be stored for > 1 day, add 0.2 g NaN3 (CAUTION - TOXIC!)
2. Blocking Procedure:
1. Immerse blot in blocking agent with agitation (i.e. a shaking or rocking table) for 1 - 2 hours at room temperature.
2. Rinse blot in PBS + 0.2% Tween 20 twice for 5 minutes each.
Blot is now ready for antibody hybridization.
ANTIBODY PROBING
1. Dilute 1° and 2° antibodies into PBS + 0.2% Tween 20 (PBST).
Note: Including blocking reagent at 0.05 - 0.1X in the antibody solutions will decrease background without significantly lowering band intensity.
The optimal dilution must be determined for each antibody. The 2° antibody may be tested on dots of 1° antibody. Dilutions can range from 1:100 to 1:10,000.
2. Incubate blocked, washed blot with 1° antibody for at least 1 hour at room temperature with agitation.
3. Wash blot four times for five minutes each with PBST.
4. Incubate blot with 2° antibody for 0.5 - 1 hour.
5. Wash blot four times for five minutes each with PBST.
6. Transfer blot to detection reagent.
DETECTION
The most popular antibody labels are isotopic (125I), HRP and Alkaline Phosphatase. 125I labeling is straightforward, and gives consistent and quantifiable results. Its drawbacks are the hazards and inconvenience which radioactive isotopes bring into the lab. Detection of 125I requires that the blot be placed against X-ray film. Upon development, the film will show bands corresponding to the position and intensity of detected antibody band.
A variety of substrates are available for both alkaline phosphatase and HRP. Protocols are given below for the most commonly used.
ALKALINE PHOSPHATASE: BCIP/NBT
Stock solutions (stable for up to 1 year):
1. 0.5g NitroBlue Tetrazolium in 10 ml 70% Dimethylformamide
2. 0.5g BCIP in 10 ml 100% DMF
3. 100mM NaCl
10mM MgCl2
100mM Tris pH 9.5
1. To prepare substrate solution: mix 100µl A, 15ml C, and then add 50µl B
2. Submerge blot (up to 150 cm2) in 15ml substrate solution. Scale up the amount of solution for larger membranes. Incubate with shaking at room temperature until desired band intensity and contrast is achieved (typically 30 minutes) depending on antibody and label activity.
3. Stop development in PBS + 20mM EDTA
Note: This stop reagent works for CIP or other eukaryotic antibodies. It does not detect Bacterial Alkaline Phosphatase.
HORSERADISH PEROXIDASE
1. Chromogenic detection with DiaminoBenzidine (DAB)
(Caution: DAB is a carcinogen - avoid skin contact. Decontaminate spent material with bleach prior to disposal)
1.
Make fresh detection solution: / 9mg DAB Tetrahydrochloride7ml 100mM Tris pH 7.6
1.5ml 0.3% NiCl2 (CoCl2 may be substituted)
6ml H2O
Filter through Whatman 1 paper
Add 15µl 30% H2O2 (or 150µl 3%)
2. Immerse blot in detection solution (up to 150 cm2/15ml) and shake at room temperature until desired band intensity and contrast are achieved. (Typically <10 minutes)
3. Stop reaction by rinsing blot with agitation in PBS.
Note: DAB development with HRP is much more rapid than the Alkaline Phosphatase/BCIP system. In addition, because the stop solution is simply rinsing away substrate, the reaction may continue for a time after "stopping". Development should be taken only up to the point where bands are acceptable and no background has yet appeared.
2. Chemiluminescent Detection using National Diagnostics' HRPL chemiluminescent detection reagent
1. Mix 7.5ml A + 7.5ml B reagents. Allow combined solution to come to room temperature.
2. Immerse blot (200 cm2/15ml) in combined A & B reagents at room temperature with shaking 1 minute.
3. Wrap blot in plastic wrap, and place in a film cassette.
4. Expose blot to X-ray film for 1 - 5 minutes, and develop film as usual.
Notes: The extreme sensitivity of HRPL can lead to high backgrounds, in which the entire blot appears as a black image on the film, even at short (< 1 minute) exposures. Background can be lowered by rinsing the blot for 1 minute in PBST after removal from the detection solution. Light emission is essentially stable for 20 - 60 minutes. Multiple exposures may be taken during this period.
STRIPPING
Western blots may be stripped and reprobed, albeit with some loss of sensitivity. Stripping generally involves the use of reducing agents such as 2-mercaptoethanol to cleave the disulfide bands which hold the antibody probes together.
Stripping solution: / 2g SDS750 µl 2-mercaptoethanol
100ml 65mM Tris HCl pH6.8
Incubate blot in stripping solution 60 minutes @ 50°C
Notes: This solution contains a high concentration of 2-mercaptoethanol: use and heat only in hood. Stripping time and temperature given are typical. Optimal values must be determined for each antibody/Antigen combination.
/ Products Related to this Discussion: /ProtoGel (30%)
30% concentrated solution of acrylamide and bis-acrylamide, 37.5 : 1 ratio. Filtered, deionized, and stabilized.
ProtoGel (40%)
Concentrated solution of acrylamide and bis-acrylamide, 37.5 : 1 ratio. Filtered, deionized, and stabilized.
ProtoGel Quick-Cast 12%
Ready-to-use gel solution for SDS-PAGE that combines the convenience of pre-cast gels with the economy of casting your own.
Tris-Glycine Electroblotting Buffer (10X)
Ultra pure transfer buffer for Western Blotting procedures. Ultra pure reagents. 18 megOhm water. 0.2 micron filtration.
HRPL Kit
Most economical and reliable horseradish peroxidase visualization system.
PBS (10X) Phosphate Buffered Saline
RNase free solution with ultra-pure reagents, 18 megohm water and 0.2 micron filtration.
ProtoBlock System
Powdered concentrate for the traditional protein blocking solution for im