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Appendix-1
5.0 Stocks of Extraction Buffer
1.0 M Tris- buffer (pH 7.5)
12.11 g of Tris was dissolved in 40 ml of ADDW and pH was adjusted to 7.5 with
6 N HCl and final volume was raised to 100 ml with ADDW, and sterilized by
autoclaving and stored at 4 oC.
5 mM Leupeptin
10.65 mg of leupeptin was dissolved in ethanol to make up the final volume of 5
ml and stored at -20 oC.
10 % Triton X-100
10 ml of triton X-100 was mixed with 90 ml of ADDW to make up the final
volume of 100 ml and stored at 4 oC.
TPCK (5 mg/ml)
5 mg of TPCK was dissolved in ethanol to make up the final volume of 1 ml and
stored at -20 oC.
Pepstatin A (2 mg/ml)
10 mg of pepstatin A was dissolved in ethanol to make up final volume of 5 ml
and stored at -20 oC.
200 mM PMSF
70 mg of PMSF was dissolved in absolute ethanol to make up final volume of 2 ml
and stored at -20 oC.
128
Preparation of 200 ml protein extraction buffer
Components
Working concentration
Volume added from stock
Tris buffer
50 mM
10 ml
Leupeptin
20 M
800 l
Pepstatin A
20 g/ml
1.66 ml
TPCK
50 g/ml
1.66 ml
Triton X 100
0.05 %
2.0 ml
Water
Final volume was raised
to 200 ml by ADDW
5.1 Preparation of Stock Solutions
5 M NaCl
58.5 g of NaCl was dissolved in 100 ml of ADDW and finally the volume was
raised to 200 ml and stored at 4 oC after autoclaving.
100 mM CaCl2
1.10 g of CaCl2 was dissolved in 40 ml of ADDW and final volume was raised to
100 ml with ADDW. The CaCl2 solution was sterilized by using 0.22 µM
disposable filters (Millipore) and stored at 4 oC.
100 mM EGTA
3.8 g of EGTA was dissolved in 40 ml of ADDW and pH of the EGTA solution was
adjusted 7.5 by using 5M NaOH. Final volume of the EGAT solution was raised to
100 ml with ADDW and stored at 4 oC after passing through 0.22 µM disposable
filters (Millipore).
4 M NaOH
16.0 g of NaOH pellet was dissolved in 50 ml of ADDW and final volume was
raised to 100 ml by adding ADDW and stored at 4 oC.
129
5.2
Preparation of Stocks for Polyacrylamide Gel Electrophoresis
Tris buffer 1.5 M (pH 8.8)
18.17 g of Tris was weighed and dissolved in 40 ml of ADDW and pH was adjusted
8.8 with 6 N HCl and final volume was raised 100 ml with ADDW. Buffer was
sterilized by using autoclave (121 oC and 15 psi pressure for 15 min) and stored at 4
o
C.
Tris buffer 1.0 M (pH 6.8)
12.11 g of Tris was dissolved in 40 ml of ADDW and pH was adjusted 6.8 with 6 N
HCl and final volume was raised 100 ml with ADDW. Buffer was sterilized by
using autoclave (121oC and 15 psi pressure for 15 min) and stored at 4 oC.
30% Acrylamide / bisacrylamide monomer stock
29.2 g of acrylamide and 0.8 g of bisacrylamide was dissolved in 40 ml of ADDW
and final volume was raised to100 ml with ADDW. Monomer solution was passed
through 0.22 M filter (Millipore) and stored at 4 oC.
Tris-glycine-SDS electrophoresis buffer (5X)
15.1 g of Tris, 94.0 g of glycine and 10 g of SDS were weighed and dissolved in
600 ml with ADDW and final volume was raised to 1000 ml ADDW and stored at
room temperature.
10 % Sodium dodecyle sulfate stock
10 g of SDS was dissolved in 100 ml of ADDW and stored at room temperature.
10 % Ammonium persulfate solution
Ammonium persulfate (APS) solution was prepared fresh. 100 mg of APS (Sigma
Aldrich, USA) was dissolved in 1.0 ml of ADDW and stored at 4 oC.
130
Composition of Laemmli buffer (2X)
Components
Tris buffer (pH 6.8)
Glycerol
SDS
-ME
Bromophenol blue
Stock concentration
100 mM
20%
4%
2%
0.2%
5.4 Composition of SDS-PAGE gel
12% RESOLVING GEL
Component
Water
30% acrylamide gel
1.5M Tris (pH 8.8)
10%SDS
10%APS
TEMED
Volume used for 20 ml (ml)
6.6
8.0
5.0
0.2
0.2
0.008
5% Stacking gel
Component
Water
30% acrylamide gel
1.5M Tris (pH 6.8)
10%SDS
10%APS
TEMED
Volume used for 10 ml (ml)
6.8
1.7
1.25
0.1
0.1
0.01
5.5 Composition of Native-PAGE gel
8% RESOLVING GEL
Component
Volume used for 30 ml (ml)
Water
14.2
30% acrylamide gel
8.0
1.5M Tris (pH 8.8)
7.5
10%APS
0.3
TEMED
0.018
131
Composition of native IEF gel (5 %)
Component
Volume used for 6 ml (ml)
ADDW
30% Acrylamide
50% NP-40
4% Ampholine
10% APS
TEMED
Composition of denaturing IEF gel (5 %)
4.394
1.0
0.24
0.3
0.06
0.006
Component
Volume used for 6 ml
ADDW
30% Acrylamide
8M urea
40% Ampholine
10% CHAPS
50% NP-40
10% APS
TEMED
Final volume
1.0 ml
2.88 g
0.3 ml
1.2 ml
0.24 ml
0.06 ml
0.006 ml
5.6 Composition of Isoelectric Focussing Gel Buffers
Buffer
Composition
Upper tank buffer
Lower tank buffer
Sample buffer
pI marker
Gel overlay buffer
20 mm NaOH
10 mm orthophosphoric acid
20% glycerol + 4% Ampholine
pH range 3 –10
8M urea
Composition of Renaturation Buffer
Component
1.0 M Tris buffer (pH 7.5)
5 M NaCl
100 mM CaCl2
BSA
100 % Tween-20
Working
Concentration
50 mM
150 mM
1 mM
1%
0.01%
132
Volume (ml) added in
100 ml
5.0 ml
3.0 ml
1.0 ml
1.0 g (w/v)
0.01 ml
Appendix 2
Fig. 1: Domain architecture of CaM-binding Sorghum bicolor Ankyrin repeat protein kinase
(SbAPK) (XP_002458488.1). Conserved domains were predicted by using Simple Modular
Architecture Research Tool (SMART) and NCBI conserved domain data base, which depicted
the presence of a 262 aa long catalytic domain (!) of protein tyrosine kinases family (aa 173 465) and low complexity region (!) starting from aa 461 – 471, three putative ankyrin repeats
domains ( ) starting from aa 48 - 79 (Domain1), aa 83 – 112 (domain 2) and aa 116 - 145
(domain 3), respectively. A putative CaM-binding domain starting at aa 171 has also identified
using CaM-binding target database.
133
1
PHOTOCLOCK 1 protein (XP_002459177.1)
CBD 1
MGEEAMDDYELHMVCYGGDDDGRVMEWESGLPGADELTPLSQPLVPPGLAAAFRIPPE PGRTLLDVHRAS 70
CBD 1
71 AATVSRLRRAPS SSSSGSGGSGSSSFAPFHPHPQAAAAAGHARGDEDADSSAAALGGGNAATTTTNTGTN 140
myb DNA-binding domain
CBD 2
RLVWTPQLHKRFVDVVAHLGIKNAVPKTIMQLMNVEGLTRENVASHLQKYRLYVKRMQGLSNE207
141 SKRPRLVWTPQLHKRFVDVVAHLGIKNAVPKTIMQLMNVEGLTRENVASHLQKYRLYVKRMQGLSNE
208 GPSPSDHIFASTPVPHSLVHEPQVPVPTSMYVAAVGSMVPMASGGQAAYHHYHHHHNATAGGGRGYPQAA 277
280
278 AYH
Fig. 2: Domain architecture of Sorghum bicolor CaM-binding PHOTOCLOCK1 protein
(XP_002459177.1). Conserved domain analysis revealed the presence of myb DNA-binding domain
(!) of SANT [switching-defective protein 3 (Swi3), adaptor 2 (Ada2), nuclear receptor co-repressor
(N-CoR), transcription factor (TF) IIIB)- and SLIDE (SANT-like ISWI (imitation switch) domain)]
family starting from aa 144, at aa 188 and two putative CaM-binding domains (!) at aa 58 – 82 and
181 – 206 with propensity score of 6 were also identified using CaM-binding target database.
134
Mitochondrial transcription termination factor (XP_002467825.1)
CBD
1
MAGTRTALALLGHDGLDSGPRPVPVVAPRRSSSSRLRVVAVALRTRPTTRLAVPGPPLPPAPAPEPVLLP
70
mTERF1
mTERF2
71 SPPVAAGAAAVLLEAGVPPADLRRAAGMCPELLSVPAEAIEAALRFLTEEAGVPAPDLPRVLRRRPRLLV 140
mTERF3
mTERF2
141 SPVAARLRPTLSSCARWGKLLPRIEFLESLGLPPRAARSMARRFPALFAYAVDGNMRPKAEYLLGAMARR 210
mTERF3
211 ADELVDFPEYFSYALATRIVPRHEACAASGVGKLPLPAMLRPGDAKFRATLASCVGSMLPRRRSPLWHAT 280
281 WVDDDDATAAAKETMV
296
Fig. 3: Domain architecture of Sorghum bicolor CaM-binding mitochondrial transcription
termination factor (XP_002467825.1). Conserved domain analysis revealed the presence
of mTERF domains (!) at aa 93 - 126, 129 – 173 and 178 – 210, respectively. A putative
CaM-binding domain (!) at aa 24 – 48 was also identified using CaM target database with
propensity score of 4.
135
S. bicolor peroxidase (SbPOD) (XP_002455406.1)
active site
1 MTTSMGSLVLLCLVSTLLFPSAVLGHPWGGLFPQFYDHSCPKAKEIVQSIVAQAVAKETRMAASLVRLHF 70
71 HDCFVKGCDASVLLDNSSSIVSEKGSNPNRNSLRGFEVVDQIKAALEAACPGTVSCADILALAARDSTSL 140
heme-binding site
141 VGGPYWDVPLGRRDSLGASIQGSNNDIPAPNNTLPTIITKFKRQGLNVVDVVALSGGHTIGMSRCTSFRQ 210
211 RLYNQTGNGMADSTLDVSYAAQLRQGCPRSGGDNNLFPLDFVTPAKFDNFYYKNLLAGKGLLSSDEVLLT 280
281 KSAETAALVKAYAADVNLFFQHFAQSMVNMGNISPLTGSQGEIRKNCRRLNNSH
334
Fig. 4: Domain architecture of Sorghum bicolor CaM-binding peroxidase
(XP_002455406.1). Conserved domain analysis showed the presence of active site (!) at
aa 60-72, a heme-binding site (!) (181-208), substrate-binding site (204-207: RCTSF),
Ca2+-binding site and eight highly conserved cystein residues (!) (C40, C73, C78, C120,
C126, C205, C237, C327), two putative CaM-binding sites at aa 50 – 70 and 252 – 275
with propensity score of 8, respectively. Putative CaM-binding domains were identified
using CaM target database.
136
Pyrabactin resistance 1 (PYR1), PYR1-like (PYL) protein (XP_002467452)
1 MVESPNPNSPSRPLCIKYTRAPARHFSPPLPFSSLIISANPIEPKAMDKQGAGGDVEVPAGLGLTAAEYE
70
SRPBCC domain
71 QLRSTVDAHHRYAVGEGQCSSLLAQRIQAPPAAVWAIVRRFDCPQVYKHFIRSCALRPDPEAGDALRPGR 140
SRPBCC domain
141 LREVSVISGLPASTSTERLDLLDDAARVFGFSITGGEHRLRNYRSVTTVSELADPGICTVVLESYVVDVP 210
211 DGNTEDDTRLFADTVIRLNLQKLKSVAEANAAAAASFVSVVPPPEPEE
258
Fig. 5: Domain architecture of Sorghum bicolor CaM-binding Pyrabactin resistance 1
(PYR1), PYR1-like (PYL) protein (XP_002467452). Conserved domain analysis of
Pyrabactin resistance 1 (PYR1), PYR1-like (PYL) protein depicted the presence of
SRPBCC (START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC) domain (!) at aa 88 -128,
two putative CaM-binding sites at aa 164 – 186 and 228 – 243 with propensity score of 7.
137
Appendix 3
Publications
Virdi, A. S., Thakur, A., Dutt, S., Kumar, S., and Singh, P., (2009). A sorghum 85
kDa heat stress-modulated protein shows calmodulin-binding properties and
cross- reactivity to anti-Neurospora crassa Hsp 80 antibodies; FEBS Letters.
583: 767-770.
Singh, P., and Virdi, A. S., (2010). Calmodulin Binding proteins: Implication in
abiotic stress adaptation. Journal of Plant Biology (Invited Review): 37 (1) 1–
17.
Virdi, A. S., Pareek, A. and Singh, P. (2011). Evidence for the possible involvement
of calmodulin in regulation of steady state levels of Hsp90 family members
(Hsp87 and Hsp85) in response to heat shock in sorghum. Plant Signal &
Behaviour 6: 3, 393-399.
138

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