Protein Extraction for myc-tagged lines
1. Grind the equivalent of 20 continuously light-grown 6 day-old seedlings in 80 µL
sample buffer.
2. Heat 95ºC for 3 min.
3. Bring to room temp, 5 min.
4. Spin down cellular debris 3 min, remove supernatant to new tube. Freeze -20ºC
for up to 1 month.
5. Load 20 μL sample per lane on acrylamide gel. (If previously frozen, heat and
spin again.)
Sample Buffer, 1X
Stock solution
Volume
1 M Tris, pH 6.8
625 µL
SDS, 20%
1 mL
Glycerol, 100%
1 mL
ß-mercaptoethanol
500 μL
aliquot and store at -20ºC
Final concentration
62.5 mM
2%
10%
5%
Proteins can be quantified by using 1 μL sample + 800 μL 1 mM Tris, pH 6.8 + 200 μL
BioRad protein assay dye reagent concentrate using 5, 10, 15, 20, & 25 μg/mL BSA
standards.
Immunoblotting
1. Remove stacking gel and equilibrate gel for 30 min in transfer buffer.
2. Measure gel and cut Whatman paper and NitroPure membrane to its size.
3. Pre-wet membrane 1 min in ddH20, then 15 min in transfer buffer.
4. Assemble transfer sandwich without any bubbles, pre-wetting sponges and
Whatman paper first in transfer buffer. Order:
Cassette top (red electrode)
Sponge
3MM Whatman paper
Membrane (NitroPure)
Gel
3MM Whatman paper
Sponge
Cassette bottom (black electrode)
5. Fill tank with transfer buffer and ice block. Buffer should cover the electrode
panels but should not touch the base of the banana plug.
6. Transfer proteins for 30 min - 1 h at 100 V with cooling, or for high MW proteins
14 V overnight.
7. Turn off power supply and disassemble the apparatus.
Immunoprobing (using Pierce kit)
1. Block membrane. Incubate 1 h at room temp (or 4ºC overnight) in 10 mL
blocking buffer, gently rocking:
2. Add 15 µL α-myc antibody (1:1000 stock kept in aliquots at -20ºC, 0.2 mg/mL).
Incubate 1 h at room temp with gentle shaking.
3. Wash 5 times in 50 mL PBS-T for 5 min each.
Visualization (in dark room)
1. Mix equal volume of luminal/enhance solution and stable peroxide solution (make
3 mL per blot).
2. Incubate blot for 5 min at room temp.
3. Remove the blot to a plastic sheet protector (press out bubbles).
4. Expose blot for 2 min to start (adjust later exposures up to 10 min).
5. Develop film and re-expose if necessary.
Blocking Buffer
PBS
1 X PBS
11.5 g Na2HPO4 (anhydrous), 80 mM
0.05% Tween 20 (or Triton X-100)
2.96 g NaH2PO4, 20 mM
5% Non-fat Dry Milk
5.84 NaCl, 100 mM
QS to 10 mL, prepare fresh
QS to 1 L, pH to 7.5, filter sterilize.
Store 4ºC.
PBS-T is PBS + 0.05% Tween-20