Fig 1.
Graphical representation of entire workflow which was followed for the isolation of chloroplast proteins from pigeon pea genotypes C. scarabaeoides and C. cajan.
Table 1.
PROTEAN®i12™ IEF system condition for IEF.
Table 2.
Comparison of total protein yield before or after modification in the extraction buffer.
Fig 2.
Two dimensional SDS-PAGE analysis of total proteins from pigeon pea genotypes C. scarabaeoides and C. cajan.
A-B) Two hundred and fifty micrograms of total proteins from pigeon pea genotypes C. cajan and C. scarabaeoides were electrophoresed on 12% SDS-PAGE gel. C-D) Hundred microgram of total proteins from pigeon pea genotypes C. cajan and C. scarabaeoides and were electrophoresed on 12% SDS-PAGE gel. E-F) Fifty microgram of total proteins from pigeon pea genotypes C. cajan and C. scarabaeoides and were electrophoresed on 12% SDS-PAGE gel. G-H) Fifty microgram of total proteins from pigeon pea genotypes C. cajan and C. scarabaeoides were electrophoresed on 12% SDS-PAGE gel (IPG strip pH gradient range is 3–10 & 4–7 for A-F & G-H, respectively).
Table 3.
Number of protein spots observed on 2D-PAGE gel.
Table 4.
Modification recommended for quality protein extraction and Isoelectric focussing using a 2-D PAGE method.
Table 5.
List of identified proteins from pigeon pea total protein after MS analysis.
Fig 3.
Ferricyanide photo reduction assay to analyse the quality of isolated intact chloroplast.
A) Rate of decrease in absorbance (A410) per unit time measured without osmotic shock. B) Rate of decrease in absorbance (A410) per unit time measured with osmotic shock.
Fig 4.
Catalase assay for determining the purity of chloroplast protein.
A) Rate of decrease in absorbance (A240) per unit time was prominent in the fraction comprised of total proteins due to the presence of in vivo high catalase activity in comparison to the fraction comprised of chloroplast proteins isolated from wild type genotype Cajanus cajan. B) Rate of decrease in absorbance (A240) per unit time was prominent in the fraction comprised of total proteins due to the presence of in vivo high catalase activity in comparison to the fraction comprised of chloroplast proteins isolated from wild type genotype Cajanus scarabaeoides.
Fig 5.
SDS-PAGE and immunoblot analysis of the proteins to determine the purity of extracted chloroplast protein.
(A) One dimensional SDS-PAGE analysis of total and chloroplast proteins from pigeon pea genotype C. cajan and C. scarabaeoides showing absence of significant number of cytosolic proteins in chloroplast protein fraction in comparison to total protein fraction. (B) Immunoblot analysis of chloroplast and total protein, using LHCb1/LHCⅡ type I chlorophyll a/b binding protein antibody, showing higher band intensity in the proteins extracted from chloroplast fraction in comparison to the one from total proteins. Equal loading of the protein was confirmed with ponceau staining and anti- LHCb1/LHCⅡ type I was used at a concentration of 1:2500.
Fig 6.
Two dimensional SDS-PAGE analysis of chloroplast proteins from pigeon pea genotypes C. scarabaeoides and C. cajan.
A-B) Around 250 μg of chloroplast proteins extracted from pigeon pea genotypes C. cajan and C. scarabaeoides were resolved during IEF over IPG strip (pH gradient range of 3–10) and then electrophoresed on 12% SDS-PAGE gel. C-D) Chloroplast protein load was reduced to 50 μg in both pigeon pea genotypes of C. cajan and C. scarabaeoides due to the lack of visually distinct spots at high concentration and were resolved during IEF over IPG strip (pH gradient range of 3–10) and electrophoresed on 12% SDS-PAGE gel, for staining of all gels short protocol of silver staining was used.