TCA

TCA-DOC

For

precipitation

of

very

low

protein

concentration

1)

To

one

volume

of

protein

solution,

add

1/100

vol.

of

2%

DOC

(Na

deoxycholat

e,

detergent).

2)

Vortex

and

let

sit

for

30min

at

4?

C.

3)

Add

1/10

of

Trichloroacetic

acid

(TCA)

100%

vortex

and

let

sit

ON

at

4?

C

(prep

aration

of

100%

TCA:

454ml

H2O/kg

TCA.

Maintain

in

dark

bottleat

4?

careful,

use

glove

s!!!).

4)

Spin

15min

4?

C

in

microfuge

at

maximum

speed

(15000g).

Carefully

discharge

supernatant

and

retain

the

pellet:

dry

tube

by

inversion

on

tissue

paper

(pellet

m

ay

be

difficult

to

see).

[OPTION:

Wash

pellet

twice

with

one

volume

of

cold

acetone

(acetone

keep

at

–

20?

C).

Vortex

and

repellet

samples

5min

at

full

speed

between

wash

es].

5)

Dry

samples

under

vaccum

(speed

vac)

or

dry

air.

For

PAGE- SDS,

resuspend

samples

in

a

minimal

volume

of

sample

buffer.

(The

presence

of

some

TCA

can

give

a

yellow

colour

as

a

consequence

of

the

acidification

of

the

sample

buffer

;

titrate

with

1N

NaOH

or

1M

TrisHCl

pH8.5

to

obtain

the

normal

blue

sample

buff

er

colour.)

Normal

TCA

To

eliminate

TCA

soluble

interferences

and

protein

concentration

1)

To

a

sample

of

protein

solution

add

Trichloroacetic

acid

(TCA)

100%

to

get

1

3%

final

concentration.

Mix

and

keep

5min

–

20?

C

and

then

15min

4?

C;

or

longer

t

ime

at

4?

C

without

the

–

20?

C

step

for

lower

protein

concentration.

Suggestion:

le

ave

ON

if

the

protein

concentration

is

very

low.

(preparation

of

100%

TCA:

454ml

H2O/kg

TCA.

Maintain

in

dark

bottleat

4?

c

areful,

use

gloves!!!).

2)

Spin

15min

4?

C

in

microfuge

at

maximum

speed

(15000g).

Carefully

discharge

supernatant

and

retain

the

pellet:

dry

tube

by

inversion

on

tissue

paper

(pellet

m

ay

be

difficult

to

see).

3)

For

PAGE-SDS,

resuspend

samples

in

a

minimal

volume

of

sample

buffer.

(The

presence

of

some

TCA

can

give

a

yellow

colour

as

a

consequence

of

the

acidifi

cation

of

the

sample

buffer

;

titrate

with

1N

NaOH

or

1M

TrisHCl

pH8.5

to

obtain

the

normal

blue

sample

buffer

colour.)

Acetone

Precipitation

To

eliminate

acetone

soluble

interferences

and

pro

tein

concentration

1)

Add

to

1

volume

of

protein

solution

4

volume

s

of

cold

acetone.

Mix

and

keep

at

least

20min

–

20?

C.

(Sugge

stion:

leave

ON

if

the

protein

concentration

is

very

l

ow).

2)

Spin

15min

4?

C

in

microfuge

at

maximum

speed

(15000g).

Carefully

discharge

supernatant

and

retain

the

pellet:

dry

tube

by

inversion

on

tissue

paper

(pellet

m

ay

be

difficult

to

see).

3)

Dry

samples

under

vaccum

(speed-vac)

or

dry

air

to

eliminate

any

acetone

resi

due

(smell

tubes).

For

PAGE-SDS,

resuspend

samples

in

a

minimal

volume

of

sa

mple

buffer.

Ethanol

Precipitation

Useful

method

to

concentrate

proteins

and

removal

of

Guanidine

Hydrochloride

b

efore

PAGE- SDS

1)

Add

to

1

volume

of

protein

solution

9

volume

s

of

cold

Ethanol

100%.

Mix

and

keep

at

least

–

20?

C.

(Sugge

stion:

leave

ON).

2)

Spin

15min

4?

C

in

microcentrifuge

at

maximum

speed

(15000g).

Carefully

disch

arge

supernatant

and

retain

the

pellet:

dry

tube

by

inversion

on

tissue

paper

(pell

et

may

be

difficult

to

see).

3)

Wash

pellet

with

90%

cold

ethanol

(keep

at

–

20?

C).

Vortex

and

repellet

sample

s

5min

at

full

speed.

4)

Dry

samples

under

vaccum

(speed

vac)

or

dry

air

to

eliminate

any

ethanol

resi

due

(smell

tubes).

For

PAGE-SDS,

resuspend

samples

in

a

minimal

volume

of

sa

mple

buffer.

TCA- DOC/Acetone

Useful

method

to

concentrate

proteins

and

remove

acetone

and

TCA

soluble

inter

ferences

1.

To

one

volume

of

protein

solution

add

2%

Na

deoxycholate

(DOC)

to

0.02%

fin

al

(for

100

?

l

sample,

add

1

?

l

2%

DOC).

2.

Mix

and

keep

at

room

temperature

for

at

least

15

min.

3.

100%

trichloroacetic

acid

(TCA)

to

get

10%

final

concentration

(preparation

of

1

00%

TCA:

454ml

H2O/kg

TCA.

Maintain

in

dark

bottleat

4?

careful,

use

glove

s!!!).

4.

Mix

and

keep

at

room

temperature

for

at

least

1

hour.

5.

Spin

at

4?

C

for

10

min,

remove

supernatant

and

retain

the

pellet.

Dry

tube

by

i

nversion

on

tissue

paper.

6.

Add

200

?

l

of

ice

cold

acetone

to

TCA

pellet.

7.

Mix

and

keep

on

ice

for

at

least

15

min.

8.

Spin

at

4?

C

for

10

min

in

microcentrifuge

at

maximum

speed.

9.

Remove

supernatant

as

before

(5),

dry

air

pellet

to

eliminate

any

acetone

resid

ue

(smell

tubes).

For

PAGE-SDS,

resuspend

samples

in

a

minimal

volume

of

sam

ple

buffer.

10.

(The

presence

of

some

TCA

can

give

a

yellow

colour

as

a

consequence

of

th

e

acidification

of

the

sample

buffer

;

titrate

with

1N

NaOH

or

1M

TrisHCl

pH8.5

to

obtain

the

normal

blue

sample

buffer

colour.)