Immunoblotting
From Diseasepedia.net
No5. ImmunoblottingSummary
Detection limit is 2-20 fmoles, or about 0.1-1 ng of a 50-kD protein
Multiple steps over 2 days (or 1 long day)
Detects antigen presence, quantity, size
Semi-quantitative to quantitative
Antigen detection dependent on denaturation-resistant epitope
In general, lower-affinity antibody may work
Caution
DTT, methanol, SDS
1. Solubilize your proteins in Lasmmli sample buffer. Final sample buffer conditions should be 2% SDS, 100 mM dithiothreitol (added fresh from a 1M stock solution, which is kept frozen at -20°C), 60 mM Tris (pH 6.8), 0.01% bromophenol blue, and 10% glycerol. Whole cells can be solubilized directly in sample buffer; purified or partially purified solutions can be diluted 1:1 with 2% sample buffer. Heat to 70°C for 10 minutes. The final protein concentration should not exceed 150 ug/well (15 ug/well for minigels).
2. Run your samples on a SDS polyacrylmide gel, remove the plates, and cut the gel to the desired size for transfer. Mark the gel to estabilish orientation.
3. Cut one sheet of nitrocellulose paper and four sheets of absorbent filter paper (Whatman 3MM or equivalent) to the size of the gel.
4. Wet the nitrocellulose membrane in distilled water. Move the membrane to soak in transfer buffer for 5 minutes. Wet the absorbent paper by soaking in transfer buffer. Transfer buffer 1 (proteins 20,000-400,000 kD) : 48 mM Tris, 390 mM glycine, 0.1% (wt/vol) SDS, 20% methanol, and distilled water to 1000 ml. Transfer buffer 2 (proteins < 80,000 kD) : 25 mM Tris, 190 mM glycine, 20% methanol, and distilled water ro 1000 ml.
5. Immerse gel, membranes, folter papers, and support pads in transfer to ensure that they are throughly soaked. Be careful to exclude air bubbles from the support pads.
6. Assemble the transfer sandwich : Support pad, 2 sheets absorbent paper, gel, filter, 2 sheets absorbent paper, support pad. Place the complete sandwich in the transfer tank with membrane closet to the positive electrode (anode, red electrode)
7. Transfer overnight at 4°C. For proteins over 100,000 kD, transfer at 28 colts for 1 hour, then at 84 volts for 14-16 hours. For proteins under 100,000 MW, transfer at 63 volts for 4-16 hours.
8. After transfer, disconnect the power supply. Disassemble the sandwich and mark the nitrocellulose membrane to retain orientation.
9. Rinse the folter several times with PBS.
10. Incubate the blot in 5% nonfat dry milk / PBS at room temperature with agitation for 2 hours. Then remove the blot from the blocking solution and wash twice for 5 minutes each in PBS.
11. Add the primary antibody solution. Use 10 ml per 15 * 15 cm blot. All dilutions should be done in 3% BSA/PBS.
12. Incubate the blot with antibody for 1 hour at room temperature with agitation. Wash the blot with four changes of PBS for 5 minutes each.
13. Add horseradish peroxidase-labeled secondary reagent. All dilutions should be done in 3% BSA/PBS. Use tissue-culture supernatants either undiluted or diluted up to 1 in 10. This yields an antibody concentration between 1 and 50 ug/ml. For polyclonal sera or ascites fluid, a dilution of 10 ul in 10 ml will normally be sufficient. If the concentration of an antibody solution is unknown, try several dilutions to determine the correct range.
14. Incubate for 1 hour at room temperature with agitation. Then wash the blot with foir changes of PBS for 5 minutes each.
15. Immediately prior to use, prepare fresh chemiluminescence reagent. Prepare only as much reagent as needed, because the reagent has a very short chelf life. Incubate with the blot for 1 minute.
16. Drain the blot and remove excess chemiluminescence reagent by blotting the edge or corner of the filter with a paper towel. Place the blot in plastic wrap to ensure a dry surface for film exposure.
17. Expose the blot to film in the dark room for 1 minute. Develop the film and determine the correct exposure time for your antigen. Exposure times may range from a few seconds to several hours.
