WESTERN BLOT PROTOCOL
In a Western blot, proteins that are separated on polyacrylamide gels on the basis of size are transferred to a membrane for detection with antibodies.

Preparation of Human Cell Extracts (per Yasu Itoh, 5/91)

1. Add 2 volumes of autoclaved H
2O for each volume of cell pellet.
2. Bring to 1 mM PMSF.
3. Sonicate on ice (1 sec/ml, 5 times at setting 5). Wait 30 seconds between each sonication.
4. Pellet debris in the microcentrifuge at 13,000 RPM for 5 minutes.
5. Dilute with water to OD
280 = 3.0. Store at -20°C. These stocks should be good for at least one year.

Preparation of Bacterially Expressed Proteins

1. Grow a 10 ml bacterial culture to saturation with the appropriate antibiotic. If IPTG induction is needed, transfer 200 µl of the saturated culture to 10 ml of L broth with antibiotic. IPTG may be added at this time, or the cells may be grown to attain exponential growth before adding the IPTG. Add 10 - 100 µl of 1 M IPTG so that the final concentration of IPTG is from 1 - 10 mM. Induce protein synthesis for 1 - 3 hours. The final yield of protein may be affected by the concentration of IPTG, the induction time, and the bacterial concentration.
2. Centrifuge at 2,500 RPM for 15 minutes at 4°C. Discard the supernatant.
3. Dissolve the pellet in 1 ml of lysate buffer. Bring to 1 mM PMSF by adding 100 mM PMSF stock (17.42 mg/ml in methanol). This solution may be kept for 1 week at -20°C.
4. Add 50 µl of a 10 mg/ml lysozyme solution made in 0.25M Tris-HCl, pH8. Make the lysozyme solution fresh each time. Do not vortex the enzyme. Shake the cells on ice for 15 minutes.
5. Transfer the cell solution to a 4 ml Falcon tube and sonicate at setting "4" for about 10 seconds on ice.
6. Transfer this lysate to microfuge tubes and centrifuge for 15 minutes on high speed (13,000 rpm) at 4°C.
7. At this point, you will need to determine if the antigen is in the supernatant or in the pellet using a Western blot or ELISA. (In the case of the recombinant 52 kD Ro/SSA, the antigenic pellet may be stored at -20°C for about 1 month or -70°C for about 3 months.)

SDS-PAGE Separating Gels (Hoeffer baby gels):

1. For 2 gels, mix in an Erlenmeyer flask in the fume hood with constant stirring under vacuum (to remove air bubbles) for 15 minutes:
6 ml lower gel buffer at room temperature (due to the SDS)
6 ml 40% acrylamide
12 ml double distilled H
20
(-- or use 7.95 ml of 30% acrylamide and 10.05 ml H20 --)
Increase the percentage of acrylamide to analyze lower molecular weight products (less than 30 kD), but continue to use a 10% stacking gel.
2. Assemble the gel holder. Wear gloves to clean the plates with methanol or 95% ethanol. Dry with Kimwipes. Assemble in a gel caster in the following order (for 2 gels):
a). wax paper
b). thick plastic plate
c). thin, flexible plastic spacer plate
d). white glass plate
e). Gray plastic side spacer
f). Clear glass plate
g). thin, flexible plastic spacer plate
h). Repeat 4-7, then clamp sides.
3. Add 0.12 ml of 10% ammonium persulfate solution and 12µl of TEMED (kept at 4°C) to the acrylamide solution prepared above. The APS solution should not be more than 1 week old.
4. Pour the acrylamide solution into the gel holder to about 1 cm above the black mark. This black mark is 3 cm from the top of the plastic holder. Add 1/2 Pasteur pipet full of H
20 saturated butanol to each plate.
5. Wait at least 45 minutes for polymerization then rinse the top of the gels with distilled H
20. These gels may be stored at room temperature for about 1 week in a sealed container with H20 soaked towels to avoid dessication of the gels.
6. Clamp in electrophoresis holder and rinse again with H
2O, then blot the top of the gel with filter paper to remove excess H2O.

Prepare 10% SDS Stacking (or upper) Gel:

1.25 ml upper gel buffer at room temperature
0.56 ml 40% acrylamide
3.2 ml H2O
(-- or 0.75 ml 30% acrylamide plus 3.0 ml H2O --)
30 µl 10% APS solution
10 µl TEMED
Load the upper gel with a Pasteur pipet and add the gel comb. Watch polymerization about every 3 minutes to see if more upper gel solution needs to be added. It should be completely polymerized within 15 minutes. Remove comb and add Tank Buffer to the top with a funnel. Remove any bubbles at the bottom of the gel. Rinse all wells with tank buffer.


Preparation of Sample:

1. Consider volumes to be loaded: For the 1 well comb, load 10 µl of protein molecular weight markers and 500 µl of sample. Use 30 µl of sample per well for the 10 well comb. If a similar gel is prepared for Coomassie blue or silver staining, additional protein needs to be loaded relative to that used for Western blots. See the Coomasie and Silver Staining protocol for details.
2. Add sample buffer to the samples:
* Molecular weight markers from Promega are at 0.5 mg/ml. These are diluted 1:2 in H2O and stored at -20°C. Before use, add 20 µl of this dilution to 10 µl of H20 and 10 µl of 4x sample buffer (or 20 µl of 2x sample buffer). Molecular weights are listed below.
* For human cell extracts add 167 µl of 4x sample buffer to 500 µl of Molt-4 extract (A
280 = 3.0) before heating. This will dilute the sample buffer to a 1x concentration.
* For bacterial cell extracts, dissolve the final antigenic pellet (or heat the bacteria) in 500 µl of 2x sample buffer, then dilute 1:10 in additional 2x sample buffer.
* For insect cells infected with baculovirus, use a lysate from 3000 cells per lane in a 10 well comb with diluted sample buffer.

# Heat samples at 95°C for 10 minutes before loading on the gel.