EXTRACTION EXPERIMENTS

on

COATED VESICLES

Ogan Gurel Harvard College

1 July 1984

Introduction and Background

preparation of assembly factor (lOOK and SOK 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 lOOK and SOK 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

4 C

30 minutes 25 psi

Pellet Manipulation

bottom 50 lamda of supernatant was always discarded

pellet was taken up in two steps:

1) stirring with closed micropipette

2) micropipetting 12 times at 150 lamda

Incubation on Ice

bath assembly

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 Iml 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 10ug,25ug, and 50ug loadings. The new CV's are on lanes 3,4,and 5 respectively.

Clathrin, lOOK, tubulin, 50K and light chains can be identified. The lOOK protein appears to be degraded into a number of bands.

ABC

o{c1 CV5 C\/~ oldC"';

,," 5). 10.\' 5,\. 12>' 25A :5 >-

~B<""

. ((f",

#.A',

CV Prep -- Mini-gel

Extraction 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 O.SM Tris-A buffer pH 7.0.

18, Ip supernatant and pellet, respectively, of control
28, 2P 30 minute incubation on ice
38, 3P 60 " " " "
48, 4p 90 " " " "
S8, SP 120 " " " "
68, 6P ISO " " " " 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.

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; ,,:.:

' .. ····ic.""·

-

Extraction A 6/21/84

Extraction 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.

18, IP 0.25 effective Tris concentration
28, 2P 0.5 M " " " (control)
38, 3p 1.0 M " " "
48, 4P 1.5 M " " "
58, 5P 2.0 M " " "
68, 6P 2.5 M " " "
Gel B was loaded incorrectllYi as such the gel was
redone as B-2 and all results for this experiment
are interpreted from this gel. Conclusions:

Extraction of all proteins (including surprisingly, tubulin) improves as Tris concentration increases. Optimal lOOK and 50K extractions occurs in 58 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.

"III.

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Extraction B

6/22/84

I

It' 25 2P 3S 3;:> 45 4? SS SP GS 6P CVs

- - - -- - -~~ ~~p~},

~

- _---------._

Extraction B-2 6/24/84

Extraction C -- pH

In this experiment, extractions in O.SM 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.

18, IP pH 6.S
28, 2P 7.0 (control)
38, 3P 7.S
48, 4P 8.0
S8, SP 8.S
68, 6P 9.0 Conclusions:

An interesting pattern may be observed in this gel. lOOK protein is sparingly extracted in basic conditions (ph 9.0) while SOK protein is more

readily extracted under such conditions and perhaps slightly less extracted at lower pH values. However, optimal extractions of both 100 and SOK occur at

pH values of 7.0 and 7.S (28 and 38 respectively).

Extractions seem to follow the pattern:

acidic

6.S lOOK

7.0/7.S

9.0 SOK

extracted

optimal extraction extracted

basic

Extraction C 6/22/84

Extraction D (regular & normalized)

-- Multiple 8pins on Buffer A

In this experiment, the effect of multiple spins on

the extraction of assembly factor was analyzed. An initial incubation in O.SM 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 lSO lambda as supernatant.

S8 and SP represent a slight modification of this theme as two spins were done with the first pellet taken up in O.SM Tris-A buffer. The diagram on the next page should clarify the procedure followed. All incubations were' for 30 minutes at room temperature.

18, lP 1 spin (control)
28, 2P 2 spins
38, 3P 3 spins
48, 4p 4 spins
S8, SP 2 spins with O.SM Tris-A buffer. The normalized gel is loaded differently to equalize the different dilutions of supernatant after taking lSO 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 lOOK 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.

c~ CVS 15

f'

IP 2S .2? 3S3? ..<is <TP S'S :s? CVS

-

Extraction D 6/25/84

is)..

G/S CVS IS

'"

-

-

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--

Extraction D (Normalized) 6/25/84

Extraction D (revised) -- Multiple Spins in O.SM Tris

This experiment consists of two parts. The first one analyzed successive supernatants from 1 to 4 spins

with a final pellet (lS,2S,3S,4S,P). Between each

spin the pellet was resuspended in O.SM Tris-A buffer (i.e. 100 lambda buffer A, 100 lambda 1M Tris-HCl pH 7.0).

The second part of the experiment involves two spins in which the first pellet is taken up i~ O.SM Tris

exclusively (200 lambda). Here too, the supernatants after each spin are analyzed. The procedures are diagrammed

on the next page.

In both cases, initial incubation is in O.SM Tris-A buffer and all incubations are for 30 minutes at room temperature.

, ,

IS ,2S ,2P supernatants and pellet (after O.SM exclusive Tris incubation). lS,2S,3S,4S,P successive supernatants after O.SM effective Tris-A buffer resuspension of pellet

Conclusions:

Successive spins do extract more assembly factor

in each supernatant and it appears that 3 spins pull off most of the lOOK and SOK proteins. Resuspension of the pellet in O.SM Ttis exclusively doesn't seem to extract any more assmbly factor than O.SM Tris-A yet it does extract slightly less Clathrin after the second spin.

It might prove interesting to try this second spin with a higher concentration of Tris which would parallel the results found in Extraction A.

Extraction D (revised) -- Multiple Spins in O.SM Tris

Procedure Diagram

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(P)

+ '0")' "B AJOb> I.Ot.-(

/~

(+»

+- tOO). ']?, A- 1.00,\ I·C"1

/\

/\

15 '

(p)

+- 2-00)' O. S ~

··25'

(.2..p I

-f ;'(0). !3.A-

Extraction D (Revised) 6/25/84

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