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Summary BASIC LABORATORY MOLECULAR SEPARATION BIOTECHNIQUES

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TITLE: INTRIDUCTION TO BASIC LABORATORY CENTIFUGATION TECHNIQUES DESCRIPTION:EXPLAINS IN DETAILS METHODS,TYPES AND PROCEDIRES OF VARIOUSE LABORATOY CENTRIFUGATION SEPARATION TECHNIQUES SUBJECTS:DEALS WITH GENERAL PROCEDURES FOR SEPARATION AND QUANTITIFY MOLECULES OF INTEREST IN THE BIOTECHNOLOGY

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EGERTONUNIVERSI
TY
BMED31
8:BIOCHEMI
CALTECHNI
QUES
LECTURENOTES
I
n troduc
tion
Biochemist
r yi sthes t
u dyofc hemi calp rocessestha ttakep laceinl i
vingorga ni
smswi t
ht heult
ima te
aimo fu nder s
tan di
n gt hen atureo fl i
fei nmo l
eculart erms .Bi ochemicals t
u di
esa rea i
dedb y
avail
ab i
li
tyo fa n al
y t
icalt e
chn i
q uest ha ta dvancet hekn owledgeo ft hen a t
urea ndr e
lati
onships
betweenb i
o logi
ca lmo lecul
essu c
ha sn ucleicac i
dsa ndprotei
n sa ndtheircell
ularfu ncti
on.
I
d enti
ficat
io no fc onstitu
ents:e xp e
r imenta ldesi
gn
Exp eri
mentsa rec a refull
yd esign ed,e xecuteda ndd ataa nalyzedtoa nswe rs peci
ficq ues
tio
n so r
hyp o
theses.Ex perimen ta
ld e
signi nvolvesan umbe rofs t
age s:
·theidenti
fi cati
ono ft hes ubj
e c
tfo re xperime nt
ali nv
esti
ga ti
o n;
·thec ri
tic
a le v
alu a
tiono fthec urr ents ta
t eofkn owl e
dge( the‘ li
terature
’)oft hec hosensubje
cta rea
notingthes tr e
ngt hsa ndwe aknes seso ft heme t
ho dol
ogiesp reviousl
ya ppl
ieda ndthen ewhy pot
he ses
whiche mer gedfr omt hestudies
;
·thefo rmula ti
ono ft heq ues
tiono rhy pot
he sistob eaddressedb yt heplannedexperime n
t;
·thec are
fu ls e
lectiono ftheb iol
o gicals ystem( s
p eci
es,inv i
voo rinv i
tro)tob eusedfo rthestudy;
·theid e
ntificationo ft hevariablet ha tist ob estudie
d;t hec o n
siderat
ionoft heo therv a
riabl
est hat
willn ee
dt ob ec ontrolle
ds ot ha tt hes electedv aria
b l
ei st heo nlyfa ct
ort hatwi lld e
termin
et he
experimentalo utcome ;
·thed esi
gno ft hee xp er
imentin cludingt hes tatist
icalanalysiso fther esul
ts,c a
r efulevalu
ati
o no f
thema teri
a lsanda p paratustob eu seda ndt hec o
n sequ
entialpo tent
ialsafetyaspectso fthestudy;
·thee xecutiono ft hee xperi
me nti ncludinga pp
r opri
atec ali
b rati
onsa ndc o
ntrols
,wi thac arefully
wr i
ttenrecor doft heo ut
comes;
·ther epl
icationo ft hee xp
e r
imen ta sn ece
s saryfo rtheu namb iguousanalysi
so ftheo utcomes;
·t hee valua ti
ono ft heo utcome si nclud i
ngt hea p p
licati
o no fa ppr
op ri
ates tatis
tica
lt es
t st o
quantit
ativ
ed atawhe reappli
cab l
e ;
·thefo rmula ti
ono ft hema inconc lusi
o nstha tcanb edr a
wnfr o mther esu
lts;
·thefo rmula ti
ono fn ewhy pothes esa ndoffu turee xper
ime ntstha temergefr omt hes t
udy




Quant
it
a t
ivemeasu
rement
a)Uni
tsofme as
urement
TheFrenchSystè
meInter
nat
io
nalďUni

s(t
heSIs
yst
em)isthea
ccep
tedc
onve
nti
onforal
luni
tsof
meas
urement.Onemoleofasubs
tan
cei
sequ
alt
oit
smo l
ecul
armassex
pre
sse
dingrams,whe
rethe

1

,molecularma ssi sthes umo fthea tomicma sse
so ft hec onsti
t uen
ta t
oms.No tethatt het e
r m
molecularma ssisp referredtot heo ldertermmo lec
u l
arwe ight.TheSIu nitofc oncent
r at
ioni s
-3
expres
s e
di nt e
rmso fmo l
esp e
rc ubicme tre(molm )( Table1 )
.I np rac
ticethisisfa rtoolargefo r
3 -
3
normallabora t
oryp urpos
esa n
dau nitb ase
do nacu b
icd eci
me ter(dm,1 0 m)i spreferred.
Themo lari
tyo fas o
lutio
no fas u
b st
anc eexpres
sesthen u mb e
ro fmo l
esoft hesubstanceinonec u
b ic
deci
met e
ro fs ol
ution
.I ti
se xpr
essedb ythes ymbolM.
I
ts houldben otedthata t
omicandmo lecularma s
sesa rebo thexp re
s s
edinDa lton
s( Da)orkiloDa lt
on s
(kDa),whe reo neDa l
tonisa natomicma ssu ni
tequalt oon etwe l
ft hoft hema sso fonea t
omo ft he
1
2
Ci sotope
.Ho we v
er,b io
chemist
sp refert ouset het er mr elativ
emo le
cularma ss( Mr).Thi si s
1
2
defin
eda st hemo lecul
arma ssofas ubstancerelat
ivet oo ne-t
we lftho fthea tomicma ssoft he C
i
s o
tope.Mrt hereforehasn ouni
ts.
Thust herelativ
emo lecula
rma sso fsodiumc hlo
rideis23( Na)p lus35. 5(Cl)i.e.58. 5,sot haton e
moleis58. 5gr ams.I fthiswasdissolvedinwa t
eran da d
ju s
t e
dt oat ota
lv ol
umeo f1d m3t hesolut
io n
wouldb eo n emo l
ar( 1M) .Bi o
logicals ubsta
ncesa r emo s
tfr equen
tlyfo unda tr el
ati
velyl ow
conce
ntrati
on sa ndini nv itr
omo de
ls y s
temst hev o
lume so fs tocks olut
ionsr egularl
yu sedfo r
experi
me nt
alp urposesarealsosmall.Thec onseque
ncei stha texperimentalsolu
tionsareu s
uallyinthe
mM,n Ma ndp Mr anger a
therthanmo lar.
Table1:SIu ni
ts-Basicuni
ts


Qu
ant
it
y SIu
nit Sy
mbo
l

Le
ngt
h Me
tre m

Ma
ss Ki
lo
gra
m kg

Ti
me s
eco
nds s

El
ect
ricc
urr
ent Amp
ere A

Te
mpe
rat
ure Ke
lvi
n K

Lu
min
ousi
nte
nsi
ty Ca
nde
la c
d

Amo
unto
fsu
bst
anc
e Mo
le mo
l


I
nscie
nceitiscri
ti
caltha
texper
iment
sandtheob
ser
vati
onsrec
orde
dar
erepea
tabl
e.
There
forequ a
nti
tat
iveobser
vati
onsaremu c
hmo r
ev a
luabl
et ha
nqual
it
ati
ve.Whenan ume
ric
al
val
ueisgi v
ent oac hara
cte
ris
ticital
lowsfordire
ctc o
mparis
onbe
tweenobs
erv
ati
onsmad
eb y

2

, di
fferentp eopleora tdifferentti
mes .
Qu a
nti
tativeme asuremen tiscoll
ecti
ngi nformationthatisnumericinn a
ture.
Qu a
nti
tatived ataha sv al
ueso fav ariabledifferi
ngi na mountinn ume r
ict ermsr a
therthanin
desc
ript
ivet erms.
Qu a
nti
tativeme t
hodsa rer es
earchte chniquesthatareu se
dt ogatherq ua
n t
itat
ived a
ta-in
formati
on
deal
ingwi thn umbersa nda nyt
hingtha tisme asurable
.St at
isti
cs,tablesandgr aphs,areofte
nu s
ed
topresentt her esul
t soft heseme thod s
.The ya r
et here
foret ob ed i
st
inguishedfromq ual
it
ativ
e
methods
.
c)Accuracy&Pr ecision
Accurac
ya n dp reci
sions houldb ec o nsi
deredwhe therc oll
ect
ingd at
ao rme asuri
ng mater
ials
.
Accuracyr eferstoho wc lo
seame asure dvalueistothet r
uev al
ue.
Preci
sio
nr eferstoho wr eproduc
ibleame a
surementis.Ify our epeatame asurementan umberof
ti
mes,itisho wc losethen umerica
lva l
u esaretoe ac
ho ther
.
d)Scie
ntificNo tati
on
Whe nr ecordinga nda n al
yzingn ume ri
cald atay ouma ye ncounte
rv eryl argen umbers(suc
ha s
987,000, 000, 000)o rv erys malln umb ers(sucha s0. 000000001 23).Si n
cet hesenumbersa r
e
di
fficul
tt owo rkwi tho renterint
oac ommo ncalcula
torweu sesci
e n
tifi
cn ot
a t
ion.Scie
nti
ficnot
ati
on
repre
sentsan umb erinthef o
llowi
ngfo rmat:
Nx1 0nwhe reN~1( N<1 0)a ndnist hen umb e
ro fspacesthedecimalpointismo v
e d
;totheleftifa
negat
ivenu mbera ndt other i
ghtifap osi
tivenumb e
r
1
1
Example:987 ,000,000, 000=9. 87x1 0a nd0. 00000000123=1 .23x1 0-9


e)Solu
tesa n
dSo lv
ents
Al i
qui
ds olut
ionisami xt
u r
eo ftwoormo resubstanceseve
nlydis
tri
b ut
edwi t
hineacho ther
.The
subs
tancewhicha c
tsastheliqu
idandcompri
sesthema jori
tyofthetotalvol
umei sc
a l
le
dt hesolv
ent
andtheo t
hersubstan
cedisso
lve
dinthesolve
ntisc a
lledthesolu
te.Typica
lly,theconcent
rati
onofa
sol
uti
oni n
dic
ateshowmu chsolu
teisdi
ssol
vedinsol
ventfo ragiv
envolume.
Forexamp l
e:Yo uhaveasolut
iont
hatis10mg/mL( NaCl/H2O)
·Thesodiumchlori
de(NaCl)i
sthesol
uteandthewa ter(H2O)isthesol
vent
·Theconcent
rati
ontell
susho wmuchsolut
e(10mg)i sdiss
olv
edineachmLo ft hesol
uti
on.
f)Concentra
tio
n s
Therearenumerouswaysofr ep
orti
ngconce
ntrati
on:
i
)Ma ss/volu
me( mg/mL).Ma sstovol
umes ol
uti
onsa recalc
ulat
edbyt heamo untofsolut
ed is
sol
ved
pervol
u meofsoluti
on.Fore xa
mple,i
fy ouha v
e10mg/mL( NaCl/H2O)s oluti
on,therearetenmg
ofsodi
umc hlor
idedis
sol
vedinonemLo fsol
ution
.
i
i)Pe r
c en
ts ol
uti
onsd e
termine
db ywe i
ght/volume.Pe rcen
t( %)sol
u t
ion
sb yweight/volu
mea re

3

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