Aplikovaná chemie a biochemie
Přednáška č. 1
Úvod
Stručný přehled:
• detekce exprese, lokalizace, syntézy,
degradace a funkcí proteinů; protilátky;
• detekce exprese na úrovni RNA; využití
siRNA pro manipulace s expresí genů;
microarrays;
• práce s DNA; využití vektorů pro
transfekci
živočišných buněk in vitro; aktivace
transkripce;
• lipidy a polysacharidy – izolace, separace a
identifikace;
• nízkomolekulární látky – separační a
analytické techniky, HPLC techniky a
modifikace;
• využití chemických inhibitorů,
• modelové organismy; transgenní zvířata.
Náplň předmětu:
• přednášky;
• seminář;
Literatura:
• Molecular Biology of The Cell (s.469-580),
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=books
•Principles and Techniques of Practical
Biochemistry
http://assets.cambridge.org/97805216/58737
/toc/9780521658737_toc.pdf
Primární literatura
Protein (1):
• identifikace (specifická protilátka; MALDITOF;
microsequencing)
• zařazení (práce s databázemi)
• regulace exprese (analýza exprese v
modelovém buněčném systému a in vivo)
• lokalizace v buňce (live cell imaging;
imunofluorescenční techniky; subcellular
fractionation)
• interagující proteiny (imunoprecipitace;
GST-pull-down; yeast two-hybrid screen)
• in vitro funkce – enzymatická aktivita?,
strukturní protein?, membránový protein?,
adaptérový protein?, receptor?;
• in vivo funkce (transgenní modely, tkáňově
specifická exprese, mutace)
Protein (2):
• funkce jednotlivých domén;
• posttranslační modifikace (fosforylace,
acetylace, glykosylace)
• regulace odborávání proteinu (ubikvitinace;
proteazomální degradace)
• DNA interakce, transkripční aktivace,
identifikace cílových genů, DNA-binding sites
(EMSA, reporter gene assays)
• manipulace s proteinem (overexprese;
dominant negative constructs, antisense
oligonucleotides, siRNA)
• proteomika
• protein v buněčném kontextu
• izolace a purifikace
SDS-PAGE a Western blotting, 2-D elektroforéza
Western blotting – transfer:
Membrány:
• PVDF
• nitrocelulóza
Western blotting – imunodetekce:
Western Blot Procedure
• Blocking
The membrane is blocked, in order to reduce non-specific protein interactions between
the membrane and the antibody. This is achieved by placing the membrane in a solution
of bovine serum albumin (BSA) or non-fat dry milk (NFDM).
• Primary Antibody
The first antibody to be applied (specific for protein of interest) is incubated with the
membrane. The antibody is diluted in a buffer solution (PBS) containing a carrier
protein (BSA or NFDM) along with some detergent. The primary antibody is specific for
the protein of interest, and, at appropriate concentrations, should not bind any of the
other proteins on the membrane.
Western blotting is used to detect a
target protein in a sample (containing a
complex mixture of proteins) by using a
polyclonal or monoclonal antibody
specific to that protein.
• Secondary Antibody
After rinsing the membrane to remove unbound primary antibody a secondary antibody
(figure 3 – green antibody) is incubated with the membrane. It binds to the primary
antibody. This secondary antibody can be linked to an enzyme that allows for visual
identification by producing vissible changes in colour, chemiluminescence or
fluorescence. An alternative is to use a radioactive label.
• Developing
The unbound secondary antibodies are washed away, and the enzyme substrate is
incubated with the membrane so that the positions of membrane-bound secondary
antibodies will emit light. Bands corresponding to the detected protein of interest will
appear as dark regions on the developed film. Band densities in different lanes can be
compared providing information on relative abundance of the protein of interest.
1.The first paper describing the use of electrophoresis to transfer proteins from a gel to
a membrane. Towbin H., Staehelin T, Gordon J. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl
Acad Sci U S A. 1979 Sep;76(9):4350-4
2.The classic paper by UK Laemmli - the most cited article in the journal's history.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature. 1970 Aug 15;227(5259):680-5.
3. The first paper to use the term western blot. Burnette WN. "Western blotting":
electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to
unmodified nitrocellulose and radiographic detection with antibody and radioiodinated
protein A. Anal Biochem. 1981 Apr;112(2):195-203
http://www.westernblotting.org/
Modifikace
•gel-shift assays
Cx43
• whole cell protein modification
assays (e.g. tyrosine phosphorylation)
Cx43
TPA
In-gel kinase assay. MBP (0.2 mg/ml),
and MBP-Csx/Nkx2.5 or MBP-HD fusion
protein (0.1 mg/ml) were incorporated into
SDS–15% PAGE separation gels. NIH 3T3
nuclear extracts (50 mg) were boiled in
SDS sample buffer and then loaded on the
gels. After SDS-PAGE, gels were washed
twice in 20% isopropyl alcohol for 30 min,
denatured with 6Mguanidine-HCl in the
buffer (40 mM HEPES [pH 7.4], 20 mM
NaCl) for 60 min, and renatured gradually
by decreasing Renatured gels were
incubated in the presence of [g-32P]ATP
(0.25 mCi/ml)–25 mM ATP in 40 mM of
HEPES (pH 8.0)–5 mM MgCl2–2mMDTT–
0.1mMEGTA at room temperature for 1 h
and washed with 5% trichloroacetic acid
and 1% sodium pyrophosphate.
Elektroforetická separace může být využita i pro
funkční analýzu proteinů:
FIG. 4. Dose-dependent increase in new protein expression in RAW 264.7 cells as determined by two-dimensional
gels. A, dose-dependent increase in new protein expression in response to organic DEP extracts; protein expression
was suppressed by NAC. B, regression analysis showing the linear correlation between extract dose and the number of
newly expressed proteins. C, Venn diagram to show the overlapping and unique expression profiles at different doses of
the DEP extract, D, list of six new proteins induced at all extract concentrations. RAW 264.7 cells were exposed to
DEP extracts at indicated concentrations, in the absence or the presence of 20 mM NAC, for 6 h before cellular
extraction and analysis of the soluble proteins by two-dimensional electrophoresis. These data were reproduced three
times, during which the variability in protein expression was 10%.
FIG. 5. Two-dimensional gel electrophoresis profile
in the presence of 50 g/ml organic DEP extract. A,
proteins that were induced 8-fold and subtracted in
the presence of NAC were selected as oxidative
stress markers that were identified by MS. Those
proteins are numbered and their identities disclosed
in Table I. B, excerpt of the two-dimensional profile
to show how above criteria led to the identification of
GAPDH as an oxidative stress marker. The top panel
shows background expression in untreated cells, the
middle panel shows increased expression by the
extract, and the bottom panel shows the subtracted
response in the presence of NAC. C, GAPDH
immunoblotting shows the subtractive expression
of this protein in crude cell lysates. See “Materials
and Methods” for experimental details. These data
were reproduced three times, during which
the variability in protein expression was 10%.
http://www.pdb.org/pdb/home/home.do
http://www.ncbi.nlm.nih.gov/sites/entrez
Protein (1):
•regulace exprese (analýza exprese v
modelovém buněčném systému a in vivo)
• lokalizace v buňce (live cell imaging;
imunofluorescenční techniky; subcellular
fractionation)
• interagující proteiny (imunoprecipitace;
GST-pull-down; yeast two-hybrid screen)
• in vitro funkce – enzymatická aktivita?,
strukturní protein?, membránový protein?,
adaptérový protein?, receptor?;