This paper presents the results of EXTRACTION EXPERIMENTS on COATED VESICLES. The goal of these experiments was to find conditions that would optimize the amount of lOOK and SOK assembly factor proteins extracted in the supernatant.
This paper presents the results of EXTRACTION EXPERIMENTS on COATED VESICLES. The goal of these experiments was to find conditions that would optimize the amount of lOOK and SOK assembly factor proteins extracted in the supernatant.
This paper presents the results of EXTRACTION EXPERIMENTS on COATED VESICLES. The goal of these experiments was to find conditions that would optimize the amount of lOOK and SOK assembly factor proteins extracted in the supernatant.
EXTRACTION EXPERIMENTS
on
COATED VESICLES
ogan Gurel
Harvard College
1 duly 1984Introduction and Background
Preparation of assembly factor (100K and 50K band
proteins) involves basically three steps:
1) a series of centrifugations and gradients
to isolate crude CV's
2) extraction (sometimes referred to as stripping)
of assembly factor from these CV's
3) a series of columns to purify the assembly
factor from the extract.
Each extraction involves incubating the CV's in
an appropriate buffer under suitable conditions and
then centrifuging to recover the supernatant. The
goal of these extraction experiements was thus to
find conditions that would optimize the amount of
100K and 50K assembly factor proteins extracted in
the supernatant.
This paper presents the results of those experiments.PROCEDURAL NOTES
Electrophoresis
5% stacking gel
10% running gel
100-120 volts
60 lamda loadings except for Extraction D
(normalized)
and Prep mini-gel
5 and 10 lamda marker loadings
Centrifugation
Beckman Airfuge at 4C
30 minutes
25 psi
Pellet Manipulation
bottom 50 lamda of supernatant was always
discarded
pellet was taken up in two step:
1) stirring with closed micropipette
2) micropipetting 12 times at 150 lamda
Incubation on Ice
bath assembly : fs
firfuge tube
A ae
Ove
Control Extraction
100 lambda CV's in buffer A with 100 lambda
1M Tris HCl buffer pH 7.0, giving an effective
Tris concentration of 0.5M.
30 minute incubation at room temperature in the
airfuge tube.CV Prep -- Mini-gel
Obtained 40mg in lml of CV's (Lowry analysis) and
diluted with 3 ml of buffer A to give 10 mg/ml.
Approximately Img (100 lambda) of CV's will be used
for each individual extraction.
Ran a minigel comparing these CV's with old markers
at loug,25ug, and 50ug loadings. ‘The new CV's are
on lanes 3,4,and 5 respectively.
Clathrin, 100K, tubulin, 50K and light chains can
be identified. The 100K protein appears to be
degraded into a number of bands.ACug
B25, g
uae AB
nn) c
old CVS CVs acc
— oS
Sd 1A Sd 125K Sh
CV Prep -- Mini-gelExtraction A -- TIME & TEMPERATURE
In this experiment extractions on ice at varying time
durations were compared with the control extraction
(see Procedural Notes). All extractions were
in 0.5M Tris-A buffer pH 7.0.
1s, 1P supernatant and pellet, respectively, of control
28, 2P 30 minute incubation on ice
3s, 3P 60" ; wo
4s, 4P 90" ‘ ce
5S, 5P 120 c 2
6s, 6P 150 " oI a
Conclusion
There appears to be no significant difference between
the six extractions. It is recommended, then, that
extractions proceed on ice as further degradation of
proteins may be prevented.25 2P 58 5° 45 4P 53 SP 6s EP CVE
Extraction A 6/21/84Extraction B -- Tris Concentration
In this experiment, extractions of varying effective
Tris concentrations from 0.25 to 2.5 M were compared
with the control extraction. All incubations were
for 30 minutes at room temperature in the airfuge tube.
1s, 1P 0.25 effective Tris concentration
2s, 2P 0.5 M a o (control)
3s, 3P 1.OM ” o o
4s, 4P 1.5 M 0 0
5S, 5P 2.0 M cI u
6s, 6P 25M " u a
Gel B was loaded incorrectlly; as such the gel was
redone as B-2 and all results for this experiment
are interpreted from this gel.
Conclusion:
Extraction of all proteins (including surprisingly,
tubulin) improves as Tris concentration increases
Optimal 100K and 50K extractions occurs in 5S which
was incubated in an effective Tris concentration
of 2M in buffer A. However, at this concentration,
most other proteins are well extracted including
tubulin, hence it might be better to go with an
extraction of 1.5M effective Tris concentration.Extraction B 6/22/84
cectone as B2 (ext a)
getExtraction B-2 6/24/84Extraction C
pH
In this experiment, extractions in 0.5M Tris-A buffer
were made at varying pH conditions from acidic to
basic values, All incubations, in this case, were
for 30 minutes at room temperature. 1M Tris buffer
was titrated with conc. HCl to give, the appropriate
pH solution.
is, 1B pH
2s, 2P
38, 3P
4s, 4P
58, 5P
6S, 6P
(control)
waers8
Conclusions:
An interesting pattern may be observed in this gel.
100K protein is sparingly extracted in basic
conditions (ph 9.0) while 50K protein is more
readily extracted under such conditions and perhaps
slightly less extracted at lower pH values. However,
optimal extractions of both 100 and 50K occur at
pH values of 7.0 and 7.5 (28 and 3S respectively).
Extractions seem to follow the pattern:
acidic 6.5 100K extracted
7.0/7.5 optimal extraction
basic 9.0 50K extracted5
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Extraction C 6/22/84
cvsExtraction D (regular 6 =~ Multiple Spins on Buffer A
normalized)
In this experiment, the éffect of multiple spins on
the extraction of assembly factor was analyzed. An
initial incubation in 0.5M Tris-A buffer was followed
by centrifugation into supernatant and pellet after
which the pellet was taken up in 200 lambda of buffer A
and re-centrifuged for 1 to 3 more times. After each
spin the pellet was again taken up in 200 lambda
buffer A after taking 150 lambda as supernatant.
5S and 5P represent a slight modification of this theme
as _two spins were done with the first pellet taken up in
0.5M Tris-A buffer. The diagram on the next page should
clarify the procedure followed. All incubations were
for 30 minutes at room temperature.
is, 12 1 spin (control)
28, 2P 2 spins
3S, 3P 3 spins
48, 4P 4 spins
5S, 5P 2 spins with 0.5M Tris-A buffer.
The normalized gel is loaded differently to equalize
the different dilutions of supernatant after taking
150 lambda after each spin.
Conclusions:
From the unnormalized gel we can see that the pellet
appears generally the same regardless of the number of
spins, although slightly less 100K appears in the
pellet after 2 or more spins. In the normalized
version of the gel we see little or no difference in
the supernatant indicating that successive spins after
taking up the pellet in buffer A are ineffective.
The revised Extraction D experiments should provide
further results and these are reviewed later.i 009 - Sb
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