Detection of Amino Acids and Proteins - Proteins and Molecular Mechanisms | BCH 455, Lab Reports of Biology

Download Detection of Amino Acids and Proteins - Proteins and Molecular Mechanisms | BCH 455 and more Lab Reports Biology in PDF only on Docsity! Detection of amino acids and proteins 1.Ultraviolet absorbance @ 280nm Recognizes Phe, Tyr, Trp, and disulfide bonds To quantitate- need molar absorbance coefficient of the protein Luckily a fully unfolded protein has same properties of absorbance close to its constituents in the aromatic range. So if you know the number of aromatics and disulfides you can calculate this coefficient. 2. Staining Commonly use Coomassie Blue- a dye which doesn’t react with protein but rather sticks to it forming a non-covalent complex. It makes the non-covalent complex using a mix of non-polar and ionic interactions; however, this is not true for all proteins. No one really knows how it works. R250: R=H G250: R=CH, OC,H (1.66) aS 2.
Gel
Filtra2on
 Size
of
a
protein
determines
its
rate
of
passage
through
a
molecular
sieve.
 Molecular
Sieve‐
small
par%cles
with
a
network
of
pores
into
which
molecules
 
of
 
less
than
some
maximum
size
can
penetrate.
 The
smaller
the
protein‐the
greater
the
probability
it
will
enter
the
internal
 
volume
of
the
par%cles.
 Pour
proteins
into
a
molecular
sieve.

Then
wash
with
a
buffer.

The
first
 
proteins
to
elute‐
TOO
LARGE
TO
ENTER
PORES.
 The
volume
of
buffer
required
to
elute
them‐
Void
Volume
(Vo)=
volume
of
 
column
outside
the
par%cles.
 Other
proteins
are
eluted
in
DECREASING
order
of
the
molecular
size.
 A
gel
filtra%on
column
is
calibrated
using
proteins
of
known
size.
 Note:
Funny
shapes
can
mess
you
up!
 3. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) Extremely popular method. Check protein movement in polyacrylamide gel electrophoresis in the presence of the detergent sodium dodecyl sulfate(SDS) Ct i et een Nat This method is related to gel filtrations in that the size of a protein is estimated by its migration through the small pores of a gel matrix. In this case the gel matrix is continuous rather than particulate, so smaller proteins move through faster. Use a set of standard molecular weight markers to compare your protein migration to those. The
eletrophore%c
mobility
(how
fast
something
moves
in
rela%on
to
charge)
of
a
 protein
is
determined
by
its
weight,
charge
and
shape.
 
Charge
and
shape
are
standardized
for
all
proteins
by
SDS
 
electrophoresis
 SDS
binds
to
proteins
(somehow),
disrupts
their
structure
and
shape,
dissociates
 them
into
polypep%de
chains
and
imposes
comparable
shapes
and
net
charge
 densi%es
on
them.

Don’t
really
know
how‐
It
just
works!!
 Protein‐SDS
complexes
have
electrophore%c
mobili%es
thru
polyacrylamide
gels
 that
are
inversely
propor%onal
to
the
logarithm
of
the
length
of
the
polypep%de
 chain.
 Compare
to
standards
















Get
your
weight!
 2.
Mass
Spectrometry
 Can
be
used
for
MW
determina%on
 and
with
Edman
degrada%on
to
help
 solve
the
primary
sequence
 3.
Enzyme
digest
 Use
chymotrypsin
to
cleave
aker
every
 
Tyr,
Phe,
Trp,
and
Leu
 Use
tryspin
to
cleave
aker
ever
Lys,
Arg
 Once
the
sequence
is
cleaved
using
one
 of
various
enzymes
or
procedures,
use
 Mass
Spec
on
the
fragments.

A
 database
is
used
to
tell
you
what
each
 fragment
is.


 **However,
most
sequencing
done
 from
gene
sequences.
 Assembly of 1° structure Coded information for the structures of proteins is contained in the genetic material of the chromosome, usually DNA, in the form of linear sequences of nucleotide bases. 3 sequential nucleotides contain the code for a single amino acid residue The genetic info of the DNA is constantly checked and edited by the cell to ensure that it is altered as little as possible-only the correct amino acid is placed in the correct place. Primary protein structure is sequer a chain of amino acids Amino Acid Gene
Structure‐
brief
background
 All
the
info
for
synthesizing
the
1o
structure
of
a
protein
is
encoded
in
the
 gene%c
material
of
the
chromosomes
which
is
double
stranded
DNA.
 Info
is
encoded
in
sequences
of
4
nucleo%des
on
one
strand:
 A‐Adenine
 C‐Cytosine
 G‐
Guanine
 T‐Thymine 
 
For
RNA:
T

U
(Uracil)
 The
sequence
of
the
other
strand
or
DNA
is
chemically
complementary
to
the
 first:
 A
pairs
with
T
 G
pairs
with
C



 
 
 
AGCCT
 
 
 
 
TCGGA
 
 
 
Form
genes
 
 
 
Code
for
proteins
 Prokaryo%c
cells
have
one
RNA
polymerase
that
transcribes
all
genes
but
its
 made
specific
for
different
genes
by
various
sigma‐factor
proteins.

Eukaryo%c
 cells
have
3
enzymes‐
RNA
polymerase
I,
II,
and
III
which
transcribes
different
 genes.
 I
and
III
genes
that
code
for
stable
RNA
 II
proteins
 Goal
is
to
get
mRNA:
 
1.
Eukaryo%c
gene
 
 
splicing
of
useless
por%ons‐introns
 
2.
Prokaryo%c
gene
 
 
no
splicing
 =MATURE
mRNA
 Amino acids couple to appropriate t-RNA molecules which adapt them and supply them in the proper sequence to the ribosome—mRNA complex where they are attached to the polypeptide being synthesized. SO 5’—3 nucleotides—3’ dictate what amino acid is added. Second iti § sition 3 7 positio ee is a \e0. C A G aS g@ i Phe Ser Tyr Cys U U Phe Ser Tyr Cys Cc Leu Ser Terminate Terminate A Leu Ser Terminate Trp G Leu Pro His Arg U Cc Leu Pro His Arg Cc Leu Pro Gln Arg A Leu Pro Gln Arg G Ile Thr Asn Ser U A Ile Thr Asn Ser Cc Tle Thr Lys Arg A Met Thr Lys Arg G Val Ala Asp Gly U G Val Ala Asp Gly € Val Ala Glu Gly A Val Ala Glu Gly G This is very important feature because we can change one amino acid in a primary sequence to determine which residues are the most important! Car analogy...