Vertical gel electrophoresis for Total soluble protein analysis by SDS-PAGE

To study the distinctiveness among the 12 different species of Chrysanthemum

genotypes on biochemical basis SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel
Electrophoresis) for protein profiling was done.
Preparation of stock solutions for SDS-PAGE:

A) Tris sucrose homogenization buffer (100 mL)

Constituent Stock Stock solution added Final concentration
Tris.Cl 1M 10 mL 0.1 M
Sucrose - 13.7 g 0.4 M
KCl 0.1 M 10 mL 10 mM
MgSO4 0.1 M 1 mL 1 mM
EDTA 0.5 M 200 µL 1 mM
PMSF 0.1 M 100 µL 1 mM
-mercaptoethnol 2% 100 µL 0.1% v/v
B) Resolving gel buffer (1.5M Tris.Cl, pH 8.8)
Tris.base : 18.18 g
Distilled water : 80 mL
pH of solution was adjusted to 8.8 with 1 N HCl and distilled water was added to make
the final volume to 100 mL.
C) Stacking gel buffer (0.5M Tris.Cl, pH-6.8)
Tris.base : 6.06 g
Distilled water : 80 mL
pH of solution was adjusted to 6.8 with 1 N HCl and distilled water was added to make
the volume to 100 mL.
D) Acrylamide & Bis-acrylamide solution (30%)
Acrylamide : 29.2 g
Bis-acrylamide : 0.8 g
Final volume was made up to 100 mL by addition of distilled water. Stir on magnetic
stirrer and warm slightly, filter using Watman paper and store at 4oC in amber bottle.
E) SDS solution (10%, w/v)
SDS : 10 g
Distilled water : 80 mL
Final volume was made to 100 mL by addition of distilled water.
F) APS solution (10%, w/v)
It was prepared fresh by dissolving 100 mg APS in 1 mL of distilled water.
G) Tris-glycine buffer (Electrode buffer) (pH 8.3)
Tris base : 3.03 g
Glycine : 14.41 g
SDS : 1g
Volume was made to 1000 mL by addition of distilled water.
H) Staining solution
Coomassie Brilliant Blue R 250 : 1g
Methanol : 500 mL
Glacial acetic acid : 200 mL
Final volume was made to 1000 mL by addition of distilled water and store in amber
bottle.
I) De-staining solution
Sodium chloride : 30 g
Distilled water : 800 mL
Final volume was made to 1000 mL by addition of distilled water.
J) Loading dye (pH 6.8)
Tris.base : 1.51 g
SDS : 2g
Glycerol : 2 mL
Bromphenol blue : 100 mg
-mercaptoethanol : 2 mL
Final volume was made to 100 mL by addition of distilled water.
Protein extraction
The total soluble protein extraction was done according to the method described
by Hammer and Murphy (1993) and electrophoresed in SDS polyacrylamide gel (Lemmeli,
1970).
1 g Chrysanthemum leaves were ground in liquid nitrogen to fine powder using pre-
chilled pestle and mortar and homogenized with 2 mL cold Tris-sucrose homogenization buffer.
The homogenate was centrifuged at 10,000 rpm at 4oC for 30 min. The total soluble proteins
present in the supernatant were used for subsequent analysis.
Gel preparation and its composition
A. Resolving gel (12%)
Acrylamide (30%) : 24.78 mL
Tris.CI (pH 8.8) : 12 mL
Distilled water : 22.38 mL
SDS (10%) : 0.3 mL
APS (5%) : 0.9 mL (freshly prepared)
TEMED : 45 µL
B. Stacking gel (6%)
Acrylamide (30%) : 2.7 mL
Tris.CI (pH 6.8) : 2 mL
Distilled water : 15 mL
SDS (10%) : 0.3 mL
APS (5%) : 0.3 mL (freshly prepared)
TEMED : 30 µL
Gel casting
These solutions were mixed and TEMED (N,N,N’,N’-tetra methyl ethylene diamine)
was added just before pouring the resolving gel mixture into the glass cassette of 2 mm
thickness and filled up to the 2.5 cm mark from the top of the glass plate. Care was taken to
avoid trapping of air bubble in gel solution. About 1 mL of distilled water was over layered on
the gel to cut the contact with air and to accelerate polymerization. Water was removed after
30 min, when gel polymerized. Stacking gel solution was then poured between the glass plates
and a comb was quickly and carefully inserted into the stacking gel to make wells of 7.5 mm
depth each.
Sample preparation
25 μL of the supernatant obtained was mixed with 25 L of loading dye in an eppendorf
and heated at 100oC for 2 min just before loading.
Loading of sample and running of gel
Comb was removed after gel polymerization. The glass cassette was then clamped to
the Electrophoresis Assembly (Genei, India). Electrode buffer was poured in both tanks. 50 µL
of sample was loaded in each well with the help of micro syringe and electrodes were connected
to power supply. Initially 1.5 mA current per well was supplied till stacking dye reaches to the
separating gel, after that 2 mA per well current was supplied till tracking dye reaches bottom
of the gel. The samples were electrophoresed at 100V for 5-6 hour.
Staining and de-staining
Slab gel was removed from gel cassette without breaking it and kept overnight in a tub
containing staining solution. Slab gel was transferred into another tray with De-staining
solution which is replaced twice for removing excessive stain and to clear back ground of
bands.
Documentation of gel, scoring the gel and cluster analysis
The gels were placed over a trans-illuminator and photographed. The presence and
absence of bands were scored to construct the similarity index. The degree of electrophoretic
similarity/diversity among the genotypes was calculated by pair wise comparison of genotypes
using (Rohlf, 1992).
Number of homologous bands
Similarity index =  100
Number of homologous bands + non - homologous bands
For the study of banding pattern, classification of different bands was also done on the
basis of total number of bands for each genotype, their thickness and intensity. For clustering
of genotypes on the banding pattern dendrogram was also prepared by Numerical Taxonomy
and Multivariate Analysis System (NTSYSpc, version 2.2) (Rohlf, 2009) (Exeter software,
USA).
Data was scored for the presence (1) or absence (0) bands. The band sharing data was
used to calculate genetic similarities based on Jaccard’s coefficient (Jaccard, 1908) and
UPGMA (Unweighted Pair Grouping Method using Arithmetic Averages) algorithm was
employed to determine the genetic relationship of the genotypes (Sneath and Sokal, 1973).
Horizontal gel electrophoresis using agarose
Agarose gel electrophoresis technique is simple, rapid to perform and also capable of
resolving fragments of DNA. Submarine gel electrophoresed unit (Genei, India) was used for
DNA analysis on agarose gel.
3.7.9.2 Reagents/stock solutions for agarose gel electrophoresis used and preparation
1. DNA loading dye (6X) (Double dye) 10 mL
Bromophenol Blue (0.25% w/v) 25 mg
Xylene cyanol FF (0.25% w/v) 25 mg
Sucrose (40% w/v) 4g
The components were dissolved in 8 mL of sterile de-ionized water. pH was adjusted
to 8.0 and volume was made to 10 mL and stored at -20°C.
2. Electrophoresis buffer (10X TAE) 1000 mL
Tris base 48.4 g
Glacial acetic acid 11.42 g
0.5M EDTA (pH 8.0) 20 mL
Components were dissolved in 200 mL of de-ionized water. Final volume was made up
to 1000 mL, autoclaved and stored at room temperature. 1X of TAE buffer was used for
working solution.
3. DNA staining solution and Ethidium Bromide (10000X)
Ethidium Bromide 10 mg
Sterile deionized water 1 mL
• Working solution for staining gel was made by dissolving 60 µL EtBr stock (10 mg/mL)
in 3000 mL of de-ionized water.
• Stock was stored at 4°C.
• EtBr being highly carcinogenic was handled while wearing gloves.
Procedure of gel electrophoresis
Horizontal gel electrophoresis unit was used for fractionating RAPD markers on
agarose gel using the following procedure:
• The open ends of a clean, dry plastic tray supplied with the electrophoresis apparatus were
sealed with tape so as to form a mold.
• The mold was set on a horizontal section of a bench.
• Agarose gel (2.5%) was prepared by dissolving appropriate amount of agarose in 1X TAE
buffer. Agarose was dissolved by heating the solution at 100oC. It is allowed to cool to
room temperature.
• Add EtBr to a final concentration of 0.5 g/mL (5 L of 10 mg/mL solution of EtBr to 100
mL gel mixture) and mix well.
• The melted agarose was poured into gel mold and a comb with adequate number of wells
was inserted to make the wells.
• After complete setting of the gel, the comb was removed carefully, the tape was removed
and the gel was mounted in an electrophoresis tank.
• A pre-run of 15 minutes at 50 Volt was given to the gel.
• For each well, DNA loading dye and DNA samples were mixed in 1:5 ratios and loaded in
the gel with a micropipette.
• Electrophoresis was done at 100 Volt for 4-5 hour in 1X TAE electrophoresis buffer.
• The gel was visualized in U.V. transilluminator (BioRad, USA) and stored in gel
documentation system.
Molecular markers data analysis:
Reproducibility of PCR assay was tested by performing duplicate reactions and at
different times by using identical genotypes and primer combinations and only reproducible
bands was recorded.
Scoring the gel and cluster analysis of banding pattern:
The RAPD bands obtained were scored as presence (1) and absence (0) of bands of
various molecular weight sizes in the form of binary matrix for each genotype-primer
combination of the twelve genotypes, considering each amplified band as a unique locus. Band
sharing data was analyzed to obtain genetic similarities based on Jaccard’s similarity
coefficient (Jaccard, 1908) among the isolates by using Numerical Taxonomy and
Multivariate Analysis System (NTSYSpc, version 2.2) (Rohlf, 2009). The SIMQUAL program
was used to calculate the Jaccard’s similarity coefficients and UPGMA (Unweighted Pair
Group Method using Arithmetical Averages) algorithm (Sneath and Sokal, 1973) was
employed to determine the genetic relationship of the twelve species of Chrysanthemum.

Jaccard’s similarity coefficients = NAB / (NAB+ NA+ NB)

Where, NAB is the number of bands shared by samples

NA represents amplified fragments in sample A
NB represents amplified fragments in sample B.
 Similarity matrices based on these indices were obtained. Correlation between the two
matrices obtained with RAPD marker was estimated by means of Mantel matrix
correspondence test (Mantel, 1967). Product moment correlation (r) obtained from this test
provide a measure of relatedness between the two matrices. In this instance, the matrix
correlation corresponds to two independently derived dendrograms. For matrix correlation of
this type, a correlation value (r) greater than 0.5 will be statistically significant at 0.01 probability
level if the number of observed taxonomic unit exceeds 15 (Lapointe and Legendre, 1992).
Similarity matrices were utilized to construct the UPGMA dendrograms. In order to
estimate the congruence among dendrograms, cophenetic matrices for each marker and index
type were computed and compared using Mantle test. Principal coordinate analysis was
performed in order to highlight the resolving power of the ordination.
All the gels were scored twice manually and independently. Band presence was
indicated by 1 and its absence by 0. All unique bands were also scored and included in the
analysis. Presence or absence of unique and shared and polymorphic bands was used to
generate similarity coefficients. The similarity coefficients were then used to construct a
dendrogram manually by UPGMA.
The analysis work was based on Jaccard’s similarity coefficient given as
Number of polymorphic bands
Similarity coefficient =
Total number of bands
Polymorphic information content (PIC)
Polymorphic information content (PIC) was calculated by applying the formulae
given by Powell et al. (1996) and Smith et al. (1997).where P is the frequency of jth allele for
marker i and the summation extends over n alleles.
1
PIC = 1 −
n
 Pi 2

Where, pi is the frequency of the ith allele.

Primer resolving power (Rp)
Primer resolving power (Rp) was calculated according to Prevost and Wilkinson
(1999) formulae:
Primer resolving power =  Ibi

Ibi = 1 − (2  0.5 − piΙ)

 Where, pi is the proportion of accessions containing the ith band and Ibi is the
informativeness of the ith band.
Diversity index (DI)
Diversity index (DI) is the expected heterozygosity and was calculated according to
Weir (1996) formulae:
1
Diversity index = 1 −
L
  pi 2
l i
Where, pi is the frequency of ith allele at the l locus and L is the number of loci. This formula
is equivalent to an average of polymorphism information content (PIC).
Discrimination power (D)
Discrimination power (D) determines the efficiency of primer on the basis of their
banding pattern. It was estimated using the formulae of Tessier et al (1999), as
D=1-C
Where, C (confusion probability) is the probability of the primer not to distinguish
between two different genotypes on the basis of their banding pattern. And for the ith pattern
of the jth primer, present at frequency pi in the set of N genotypes, the confusion probability
Ci is:
( Npi ) −1
Ci = pi 
N −1

Where, pi = Frequency of banding pattern

N = Total number of genotypes
For jth primer the confusion probability Cj, is equal to the sum of different Ci for all the I
pattern generated by the primer and, thus the discrimination power of the jth primer is
equal to:
l
( Npi ) −1
Dj = 1 −  p i 
N −1
i =1
Discriminating power (DL)
As N tends towards infinity, the limit of Dj, equals to Discriminating power (DL):
l
lim (Dj) = DL = 1 −  pi 2
i =1
Thus, DL is an extension of polymorphic information content (PIC) available from
different banding patterns generated by the primer.