Biology Reference
In-Depth Information
A, 10 mL Solution B, 0.0147 g CaCl 2 , 0.0197 g MnCl 2 , and
0.0203 g MgCl 2 and adjust the volume to 100 mL.
4. Elution buffer (Lectin): binding buffer plus 0.5 M
α
-methyl- D -
mannopyranoside. Dissolve 9.71 g
α
-methyl- D -mannopyranoside
5. 100 mM Ammonium bicarbonate (Na 2 CO 3 ): dissolve 1.05 g
Na 2 CO 3 in water and adjust the fi nal volume to 100 mL.
6. Resuspension buffer: 8 M urea, 100 mM Na 2 CO 3 . Dissolve
0.961 g urea in 2 mL 100 mM Na 2 CO 3 . Prepare this solution
fresh immediately prior to use.
7. 2 M Dithiothreitol (DTT): dissolve 0.154 g DTT in 100 mM
Na 2 CO 3 , adjust the fi nal volume to 500
in 100 mL binding buffer.
μ
L, divide into small
L) and store at −80 °C until use.
8. 257 mM Iodoacetamide: dissolve 0.023 g iodoacetamide in
100 mM Na 2 CO 3 and adjust the fi nal volume 500
aliquots (e.g., 5
μ
μ
L. Prepare
this solution fresh immediately prior to use.
9. 1 M NaOH: dissolve 4 g NaOH in water and adjust the volume
to 100 mL.
10. 30 % Acetic acid: mix 30 mL glacial acetic acid with 70 mL
water. This solution should be prepared in a fume hood to
avoid potentially toxic fumes.
11. Porous graphitic Carbon (PGC) wash solvent: 5 % acetonitrile,
0.1 % formic acid (v/v) in water. Mix 5 mL acetonitrile and
100
L formic acid and adjust the volume to 100 mL with
water. This solution should be prepared in the fume hood.
12. PGC Elution solvent: 50 % acetonitrile, 0.1 % formic acid (v/v)
in water. Mix 50 mL and 100
μ
L formic acid and adjust
the volume to 100 mL with water. This solution should be
prepared in the fume hood.
μ
3
Methods
1. Weigh out 3 g of plant material (fresh or fl ash frozen in liquid
nitrogen and stored at −80 °C). Replicated biological samples
should also be considered, depending on the experimental
goals.
3.1 Tissue Collection
1. Powder the samples in liquid nitrogen using a pestle and
mortar and add a tenth mass of polyvinylpolypyrrolidone
(PVPP; 1 g/10 g fresh weight) to help remove phenolic
compounds.
2. Homogenize the material in three volumes of extraction buffer
(45 mL) with a tissue homogenizer (e.g., Polytron, Kinematica)
for 15 s, and then fi lter with Miracloth (Calbiochem). Shake the
crude extract at 5 rpm on a rocking platform for 2 h at 4 °C.
3.2 Protein
Extraction
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