Berglund:Western Blot

Emily Goers Western Blotting for Semi-dry transfer 1)	Run SDS PAGE like usual, remove stacking gel 2)	Prep while gel is running (or before-hand): cut out 2 pieces of blotting paper and 1 piece of nitrocellulose membrane the same size (or slightly larger) as the SDS gel. Soak these three pieces in 1x transfer buffer while gel is running. The blotting paper can be messy, so I soak it separately. 3)	Assemble on bottom of semi-dry transfer apparatus: 1 wet blotting paper then nitrocellulose. Put a few drops of 1x transfer buffer on the nitrocellulose and carefully roll out any bubbles using a small glass test tube.  Place gel on nitrocellulose, roll out bubbles gently.  Place the second piece of blotting paper on top. 4)	Run 1 SDS PAGE gel at 35 mA, 2 gels at 70 mA etc. for 1.5 hrs. 5)	Carefully disassemble and clip one corner of the nitrocellulose to mark which side the protein was transferred to. You can also carefully write in pencil on the nitrocellulose. 6)	Optional: Ponceau stain- stains blot for protein (to make sure the transfer was successful) temporarily. Pour on Ponceau, swirl briefly, pour Ponceau back into bottle (re-usable). Rinse with DI water until bands appear. When finished viewing, rinse thoroughly with water and then TBST once. The blocking solution has TBST in it as well, so don’t worry if there is still some residual stain on the blot. 7)	Incubate nitrocellulose in clean, small container with just enough blocking solution to cover the blot for 15 min at RT to O/N at 4 degrees C. 8)	Primary antibody: Pour off blocking solution and make primary antibody solution in blocking solution. Incubate blot with Primary antibody for 1hr (minimum) at RT to O/N at 4 degrees C. Pour off, wash 3-4 times with 20-30 mL of TBST for 5-15 min each at RT. You want to make sure all free primary is rinsed away so it does not bind up the secondary antibody. 9)	Incubate at RT with secondary for 45 min to 1 hr. Secondary is also made in blocking solution. 10)	Detect using either ECL detection kit from Pierce (?) or out in-house detection system (mix 4 reagent together day of use, much cheaper). Place blot on top of saran wrap, layer 1-5 mL of detection reagent on top of blot, make sure blot is fully covered. Let react for about 5 min. pour off excess (let drip off) and transfer blot to clean, dry saran wrap. Wrap up well so no detection reagent leaks out on to film (not good). Place glow in the dark dots on saran wrap about 2 inches away from blot. Expose within 10 min of exposing blot to detection reagent. 11)	Expose blot to film (keeping it in saran wrap) for 10 sec to 10+ min in dark room. Develop film. ☺ 12)	NOTES- can dry out blot and store if you want to strip and re-probe later. Keep everything you use really clean and well-rinsed. The nitrocellulose can soak up junk easily. If you have a lot of background, wash more thoroughly after the secondary antibody and/or incubate blot with secondary for less time. Rodger knows the details on the in-house detection system. Buffers/Reagents 1x Dry Transfer Buffer: for 1L: 5.8 g Tris base, 2.9 g glycine, 200 mL MeOH, 0.375 g SDS, water to 1L TBST: 50 mM Tris-HCl (or Tris-base) pH 7.5, 150 mM NaCl, 0.1 % Tween-20 and water Blocking solution: TBST + 4-5% non-fat dried milk. Store at 4 degrees C, I would only keep around for 1 week max.