Technical Report No. 30

TECHNIQUES FOR ELECTROPHORESIS OF NAWAIIAN DROSOPHILA

W . W . M. S te iner and W . E. Johnson*

Department of Genetics University o f Hawaii

Honolulu, Hawaii

*Present Address : Department o f Biology

Western Michigan University Kalamazoo, Michigan

ISLAND ECOSYSTEMS IRP

U. S . International B io log ica l Program

November 1973

ABSTRACT

In t h i s report, we have described the equipment, techniques and chemical

so lut ions necessary t o do electrophoresis on Hawaiian Drosovhila and some

other organisms. Such techniques are useful i n analyzing genetic re lat ionships

and population structure.

TABLE OF CONTENTS Page

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

INTRODUCTION m . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

EQUIPMENTPREPARATION . . . . . . . . . . . . . . . . . . . . . . . . 1

. . . . . . . . . . . . . . . . . . . . . . GEL AND SAMPLE PREPARATION 4

. . . . . . . . . . . . . . . . . . . GEL AND ELECTRODE BUFFER SYSTEMS 14

. . . . . . . . . . . . . . . . . . . . . . THE USE OF ACRYLAMIDE GELS 18

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . BIBLIOGRAPHY 20

TABLE LIST OF TABLES

Page

. . . . . . . . . . . . . . . . . . . . 1 Gel and Bridge buffers 3

2 Buffer systems and t h e i r r e spec t ive p ro te in s t a i n s . . . . . . 6

. . . . . . . . . . . . . . . . . . . . . . . . 3 S t a i n s o l u t i o n s 9

. . . . . . . . . . . . . . . . . . . . . . . . 4 Stock solu t ions 15

F IGURE LIST OF FIGURES

Page

. . . . . . . . . . . 1 Design f o r a double-gel e l ec t rode chamber 2

2 Diagrammatic representa t ion of g e l hooked up t o e lec t rode

chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

INTRODUCTION

I n t h e fo l lowing pages we have at tempted t o assemble t h e techniques c u r r e n t l y

i n use i n t h e l a b of D r . H. L. Carson f o r s tudying evolu t ionary and g e n e t i c

events a t t h e molecular l e v e l i n t h e Hawaiian Drosophila. Most of t h e s e tech-

niques a r e a v a i l a b l e from a v a r i e t y of sources . They have been modified and

compiled f o r use i n ana lyz ing g e n e t i c r e l a t i o n s h i p s and popula t ion s t r u c t u r e s

i n t h e s e unique organisms by t h e above au tho r s . They a r e presen ted h e r e t o

provide a handy r e f e r ence source f o r t hose i n t e r e s t e d i n apply ing t h e techniques

t o problems of s i m i l a r i n t e r e s t . We have found f o r t h e most p a r t t h a t t h e s e

techniques a r e a l s o a p p l i c a b l e t o e x o t i c Drosophi l ids ( i . e . 2. simulans,

D. m e l a n o ~ a s t e r , 2. mercatorum), va r ious land s n a i l s , some c r a b s p e c i e s (Coenobita - and Calc inus spp.) and spec i e s of t h e b e e t l e family Cerambicidae.

We have a l s o used t h e s e techniques t o examine p l a n t s of t h e genera

Metrosideros and Acacia. The techniques a r e l a r g e l y i napp l i cab l e t o t h e s e t r e e

s p e c i e s a l though some p r o t e i n s ( i . e . phosphoglucomutase, phosphoglucose isomerase,

e s t e r a s e , l euc ine aminopeptidase and a c i d phosphatase) were demonstratable i n

Acacia h.

A b ib l iography i s included f o r those wishing t o i n v e s t i g a t e t h e background,

development and uses of t h e techniques of e l e c t r o p h o r e s i s .

EQUIPNENT PREPARATION

Before sample p repa ra t i on , every e f f o r t should be made t o have a l l t h e

requi red s o l u t i o n s and equipment prepared, a s most p r o t e i n s a r e i n s t a b l e a t room

temperature.

The e l e c t r o p h o r e s i s appara tus c o n s i s t s of 2 e l e c t r o d e chambers ( s ee FIG. 1)

con ta in ing a buffered e l e c t r o l y t e s o l u t i o n from TABLE 1, an e l e c t r i c a l power

source ( i . e . a Hea thki t model IP-17 power supply) and t h e molded g e l (descr ibed

b a s e p l a t e (18%" x 12")

male j a c k t o connec t chamber e l e c t r o d e t o power s u p p l y

_A ---7

,&plexiglas b lock (%I1 X %" X %I1) t o hold p l e x i g l a s rod

p l e x i g l a s r o d d i n g (%If x 17") t o s u p p o r t 18-20 gauge p l a t i n u m

I w i r e (chamber e l e c t r o d e )

6 s i d e p l a t e (18I.l x 2")

\ e n d p l a t e (2%" x 2") ,

FIG. 1. Design f o r a d o u b l e - g e l e l e c t r o d e chamber. The chamber w i l l hold two 1 9 . 5 x 1 7 . 5 cm g e l molds p laced s i d e by s i d e . A l l p l e x i g l a s p i e c e s a r e of % i n c h s i z e and may be o rde red p r e c u t from l o c a l d e a l e r s ; however, s i z e s must be s p e c i f i e d i n i n c h e s . E thy lene D i c h l o r i d e may be used t o cement t h e chamber t o g e t h e r .

TABLE 1. Gel and Bridge buffers. A l l water used should be d i s t i l l e d and deionized.

Buffer Sys tem t Y Pe PH Chemical Ingredients

A ge l 8.9 0.076 M Trizma Base (9.21 g/L) 0.005 M C i t r i c Acid-H20 (1.15 g/L)

electrode 8.7 100 m l 1 M NaOH 0.299 M Boric Acid (18.517 g/L) 900 m l d i s t i l l e d deionized H20

ge 1 8.5 0.074 M Trizma Base (9.0 g/L) 0.008 M C i t r i c Acid0H20 (1.7 g/L)

electrode 8.6 0.063 M NaOH (2.5 g/L) 0.228 M Boric Acid (14.1 g/L)

ge 1 8.5 0.074 M Trizma Base (9.0 g/L) 0.008 M C i t r i c Acid*H20 (1.7 g / ~ )

electrode 8.1 0.687 M Trizma Base (166.4 g/L) (stock solution) 0.157 M C i t r i c Acid-H20 (66.0 g/L)

Note: cathode t ray ; d i l u t e stock 1:l (v/v) anode t ray; d i l u t e stock 2:3 (v/v)

ge 1 8.5 0.015 M Trizma Base (6.2 g/L) 0.008 M C i t r i c AcideH20 (1.6 g/L)

electrode 8.1 0.029 M LiOH (1.2 g / ~ ) 0.192 M Boric Acid (11.89 g/L)

i n t h e next s e c t i o n ) . The g e l s e rves a s a br idge between t h e e l e c t r o d e chambers

(FIG. 2). The e l e c t r i c a l c u r r e n t s e r v e s t o s e t up a charged f i e l d i n t h e g e l i n

which t h e p r o t e i n molecules migrate and s e p a r a t e depending on:

(a) t h e number of p o s i t i v e ve r sus nega t ive ly charged amino a c i d s p re sen t

i n t h e p ro t e in ;

(b) t h e s i z e and conf igu ra t ion of t h e molecule wi th r e s p e c t t o t h e pore

s i z e of t he g e l mat r ix ;

(c) t h e i o n i c and s t e r i c f o r c e s c r ea t ed by the bu f fe r system and

s p e c i f i c pH;

(d) o t h e r , unknown f a c t o r s ,

Each e l e c t r o d e chamber con ta ins a platinum e l ec t rode (18-20

wire) and w i l l hold 2 g e l s . About500 m l of e l e c t r o l y t e is p l ac

its

platinum

each

chamber. The g e l i s then connected t o t h e e l ec t rode chambers v i a sponges

( D u ~ o n t , Wilmington, Delaware; 26.7 x 20 x 0.2 cm) trimmed t o 17.5 x 12 x 0.2 cm.

FIG. 2 demonstrates t he complete gel/charnber system f o r h o r i z o n t a l e l e c t r o -

phores i s .

The appara tus may be cooled t o prevent overheat ing by p lac ing i t i n a

0 c o n t r o l l e d environment ( 4 C) o r by p l ac ing i c e t r a y s on t a p of t h e g e l .

GEL AND SAMPLE PREPARATION

Gels a r e prepared using the app ropr i a t e g e l bu f fe r f o r t h e p r o t e i n system

under a n a l y s i s ( see TABLES 1 and 2) . An 11% (w/v) s o l u t i o n of e l e c t r o s t a r c h

(Otto H i l l e r , Madison, Wisconsin) i s heated w i t h continuous s w i r l i n g i n a 1000 m l

Erlenmeyer f l a s k u n t i l a v igorous ly b o i l i n g , c l e a r , viscous s o l u t i o n i s obtained.

The s t a r c h s o l u t i o n i s then degassed t o remove a i r bubbles v i a an a s p i r a t o r

u n t i l l a r g e bubbles form a t t h e s u r f a c e of t h e s t a r c h so lu t ion , about 45 seconds.

CATHODE - c u r r e n t flow -> ANODE

power source

g l a s s p l a t e

connect ing sponge

e l e c t r o d e bu f fe r e i n s e r t ion

e l ec t rode

double-gel e l e c t r o d e chamber, endview

g e l mold

FIG. 2 . Diagrammatic r e p r e s e n t a t i o n of g e l hooked up t o e l e c t r o d e chamber. Most p r o t e i n s w i l l migrate i n the d i r e c t i o n of c u r r e n t flow i f the g e l and e l e c t r o d e buf fers a r e of bas i c

pH. See t e x t f o r d e t a i l s .

- 6 -

TABLE 2. Buffer systems and their respective protein stains.

number System Milliamps gel slice Protein Stain of loci

per gel number encoded

Note: These last 6 systems

,+ -esterase, -esterase (Es) Leucine Aminopeptidase Alkaline Phosphatase Phosphoglucomutase Xanthine dehydrogenase

and/or Alcohol dehydrogenase

and/or Octanol dehydrogenase

or Acid Phosphatase

Acid Phosphatase Aldehyde Oxidase Any stain from System A

Isocitrate dehydrogenase and/or

Hexokina se Malate dehydrogenase

and/or Malic enzyme L$ -glycerophosphate

dehydrogenase and/or

Alcohol dehydrogenase Glutamate Oxaloacetate

Transaminase

(LAP) (AP) @em) (Xdh)

(Adh)

( O W

(Acph)

(Acph) (A ldox)

(Idh)

(Hk) (Mdh)

(ME)

('A -gpdh)

(Adh)

(GOT) Glyceraldehyde-3-phosphate

Dehydrogenase (G-3-pdh) or

Phosphoglucomutase ( Pgd

General Protein (G P) Any stain from Systems A or B

Adenylate Kinase (Adk) Phosphoglucose Isomerase (PGI) Amylase (Amy) Glucose-6-phosphate

dehydrogenase (G-6-pdh) and/or

6-Phosphogluconic dehydrogenase (6-Pgdh)

Aldolase ( A m

sometimes work on System C and are in need of further refinement for analysis of Hawaiian Drosophila proteins.

This s o l u t i o n i s then poured i n t o a p l e x i g l a s mold of a s i z e dependent upon t h e

e x t e n t of t h e i n v e s t i g a t o r ' s a n a l y t i c a l d e s i r e s . Usually t h i s i s a 19.5 x 17.5 x

1 cm mold (approximate volume of cooked and degassed s t a r c h s o l u t i o n = 425 ml)

which w i l l y i e l d , upon s l i c i n g i n a h o r i z o n t a l p lane , f ive 2cmthick g e l s l i c e s

f o r s t a i n i n g a f t e r e l ec t rophores i s . However, 19.5 x 17.5 x 6 cm molds

(volume = 325 m l , y i e l d t h r e e 2 cm t h i c k g e l s l i c e s ) and 19.5 x 17.5 x 3 cm

molds (volume = 220 m l , y i e l d one 2 cm t h i c k ge l ) may be used when l e s s than 5

s l i c e s a r e d e s i r e d which w i l l enable conserva t ion of s t a r c h and o t h e r chemicals.

The g e l is then cooled a t room temperature f o r 1 hour, covered wi th s a r a n

wrap t o prevent excessive dehydrat ion, and cooled f o r another 2-3 hours ( t h e

l a t t e r per iod may be hastened by p lac ing t h e g e l i n a r e f r i g e r a t o r f o r h a l f a n

hour a f t e r covering with the s a r a n wrap). A v e r t i c a l s l o t is then made 2 inches

from one end of t h e cooled g e l w i th a s a f e t y r azo r blade and a s t ra ight -edged

r u l e r a s a guide. The samples w i l l be placed i n t h i s s l o t p r i o r t o e l e c t r o -

phores i s . About 25-28 samples can be placed on a s i n g l e g e l . The g e l should

be covered wi th sa ran wrap during e l e c t r o p h o r e s i s a l so .

The samples a r e prepared a s fol lows: Whole organisms ( i .e . i n s e c t s ) o r

t i s s u e samples a r e placed i n a nylon block conta in ing 4 rows of n ine % inch

h o l e s about 1 / 3 of an inch deep and s l i g h t l y beveled a t t h e top. A nylon peg

(2 inches long, % inch i n diameter) i s then i n s e r t e d i n t o t h e holes and used t o

homogenize the samples. The peg may be a t t ached t o a multi-speed d r i l l - l i k e

appara tus t o f a c i l i t a t e homogenization. (Samples may a l s o be ground by hand on

a 9-hole spo t p l a t e using a hand-held g l a s s rod wi th a rounded end.) I n e i t h e r

case , 1 drop of d i s t i l l e d water o r buf fered g e l s o l u t i o n (TABLE 1) i s added t o

t he sample p r i o r t o homogenization.

A f t e r homogenization, t he homogenate i s soaked i n 9 x 4 mm paper wicks

(Whatman #3) . These wicks are then placed i n t h e sample s l o t i n t he 1 cm t h i c k

g e l f o r e l ec t rophores i s . I n most ca ses , 2 wicks may be obtained per sample.

For g e l s of l e s s e r th ickness , wicks correspondingly s h o r t e r than 9 mm must be

used.

When a t t a c h i n g t h e g e l t o t h e e l e c t r o d e chambers v i a t h e sponge connectors

(FIG. 2 ) , we f i n d i t u s e f u l t o p lace a g l a s s p l a t e (17 x 19 cm) on t o p t h e

sponges and g e l . The p l a t e f a c i l i t a t e s sponge con tac t wi th t h e g e l media.

A f t e r e l ec t rophores i s , t he power i s turned o f f and t h e g e l removed from

t h e e l e c t r o p h o r e s i s apparatus . The ends of t h e g e l , where the sponge connectors

overlapped, a r e then c u t o f f . Next, t h e paper wicks a r e removed and t h e g e l and

back s l i c e ( t h a t narrow p iece of g e l between t h e ca thodal sponge and the s l i c e

f o r t h e sample wicks) a r e placed upon a 1.5 mm c u t t i n g guide. A l eng th of 4

pound t e s t nylon f i s h i n g cord i s used t o s l i c e t h e g e l h o r i z o n t a l l y i n t o f i v e

1.5-2 mm t h i c k g e l s l i c e s . These s l i c e s a r e then placed i n i n d i v i d u a l s t a i n i n g

boxes (#P401, Vlcheck p l a s t i c s , Middlef ie ld , Ohio). Each s l i c e can then be

s t a ined f o r a p a r t i c u l a r p r o t e i n according t o t he s p e c i f i c a t i o n s i n TABLES

2 and 3.

Care should be taken t o inc lude t h e back s l i c e f o r s t a i n i n g when a new

enzyme o r a new organism i s being examined a s some p ro t e ins w i l l migra te

ca thoda l ly i n s t e a d of anodal ly i n a n e l e c t r i c f i e l d w i th a bas i c pH. Genera l ly

speaking, a l coho l dehydrogenase, malate dehydrogenase and ac id phosphatase have

l o c i w h i c h migra te very near t o t h e po in t of o r i g i n i n Hawaiian Drosophila.

When s t a i n i n g is f in i shed , t h e s t a i n can be a s p i r a t e d o f f and discarded. A

few s t a i n s may be saved and used over a f t e r f i l t e r i n g ( i .e . those con ta in ing

TPN o r DPN) a t t h e d i s c r e t i o n of t h e experimenter. Although t h i s can c u t chemical

c o s t s by a s much a s one-quarter , c a r e should be exerc ised a s some s t a i n s have

ing red ien t s which a r e s e n s i t i v e t o temperature. Note t h a t a l l g e l s should be

s t a ined i n t h e dark whenever p o s s i b l e a s some s t a i n s a r e photosens i t ive .

TABLE 3. S t a i n s o l u t i o n s . A l l water used should be d i s t i l l e d and deionized a s some metals w i l l i n h i b i t enzyme a c t i v i t y . See TABLE 4 f o r o t h e r s o l u t i o n s used below, * = pho tosens i t i ve s t a i n .

P ro t e in Rat ing Technique

Acph 2 Presoak g e l 30 minutes i n Borate 5.0 ( s o l u t i o n 10). Discard so lu t ion . To 50 m l of pH 5.0 b u f f e r ( s o l u t i o n 8) add: 50 mg sodium- -'A -napthy1 ac id phosphate 100 mg Polyvinyl Pyrrol idone 30 mg F a s t Blue RR With f i l t e r i n g , pour over g e l and incubate f o r 3 hours a t 3 7 O ~ , Remove s t a i n and add wash ( s o l u t i o n 18).

Adk*

Aldox

To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 5 m l MgC12 ( s o l u t i o n 16) 200 mg Glucose cq -D+-glucose) 40 mg ADP 1 m l MTT ( s o l u t i o n 20) 1 m l TPN ( s o l u t i o n 21) 40 u n i t s Glucokinase 3 m l G-6-pdh ( s o l u t i o n 23) . Pour over g e l and incubate a t 3 7 ' ~ f o r 30 minutes. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate f o r 2 hours more. Remove s t a i n and add wash ( s o l u t i o n 18).

2 To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 2 m l Isopropyl Alcohol (2-propanol) 1 m l MTT ( s o l u t i o n 20) 2 m l DPN ( s o l u t i o n 22)- Pour over g e l and incubate at 37OC f o r 30 minutes. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate f o r 1 hour more. Remove s t a i n and add wash ( s o l u t i o n 18). NOTE: This enzyme may be s t a i n e d wi th Xdh, Odh o r o(-gpdh. -

3 To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 0.25 m l of Benzaldehyde 1 m l MTT ( s o l u t i o n 20) . Pour over g e l and incubate a t 3 7 ' ~ f o r 15 minutes. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate 30 minutes more. Remove s t a i n and add wash ( s o l u t i o n 18).

3 To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 200 mg NaqFructose-1, 6-diphosphate 1 m l MTT ( s o l u t i o n 20) 2 m l DPN ( s o l u t i o n 22) 50 u n i t s glyceraldehyde-3-phosphate dehydrogenase. Pour over g e l and incubate a t 37OC f o r 25 minutes. Add 0.5 m. PMS ( s o l u t i o n 19) and incubate 1 hour more. Remove s t a i n and add wash ( s o l u t i o n 18).

TABLE 3. Continued.

P r o t e i n Rat ing Technique

3 Th i s p r o t e i n must be run on Acrylamide ge l s . To 50 m l Trizma 7.2 ( s o l u t i o n 14) add : 1 gram s t a r c h 150 mg NaC 1. Pour over g e l and incuba t e a t room temperature f o r 1 hour. Pour o f f and add a 0.01 M s o l u t i o n of Potassium Iod ide f o r 30 minutes. Bands a r e c l e a r on a dark background and w i l l f ade qu ick ly .

2 Presoak g e l 30 minutes i n Trizma 10.6 ( s o l u t i o n 15). Discard so lu t ion . To 50 m l of g e l b u f f e r (System A, TABLE 1) add: 1 drop MgC12 ( s o l u t i o n 16) 10 drops MnC12 ( s o l u t i o n 17) 100 mg Polyvinylpyr ro l idone 50 mg Na-UIC -napthy1 a c i d phosphate 50 mg F a s t Blue RR. Pour over g e l and incuba t e f o r 3 hours a t 37'~. Remove s t a i n and add wash ( s o l u t i o n 18).

To 50 m l of Nap04 A ( s o l u t i o n 6) and LO m l of NaP04 B ( s o l u t i o n 7) add: 50 m l H20 1 m l ,2 -napthy1 a c e t a t e ( s o l u t i o n 4 ) 1 m l .-5 -napthy1 a c e t a t e ( s o l u t i o n 5) 4 m l n-Propyl Alcohol. Pour 20 m l on g e l and incuba t e a t room temperature f o r 15 minutes. Add 50 mg F a s t Blue RR t o remaining 30 m l , shake and f i l t e r on to g e l . Incuba te a t 37OC f o r 45 minutes. Remove s t a i n and add wash ( s o l u t i o n 18).

1 Th i s s t a i n works b e s t i n Drosophi la la rvae . Pour 50 m l of CBB ( s o l u t i o n 27) over ge l . Incubate a t room temperature f o r 3 hours, t hen pour o f f and save f o r reuse. Rinse g e l w i th wash ( s o l u t i o n 18) a t 30 minute i n t e r v a l s u n t i l g e l i s c l ea red of background s t a i n .

3 To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 50 mg of Glucose-6-phosphate 1 m l MTT ( s o l u t i o n 20) 1 m l TPN ( s o l u t i o n 21) Pour over g e l and incuba t e a t 37OC f o r 30 minutes. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate f o r 2 hours more. Remove s t a i n and add wash ( s o l u t i o n 18). NOTE: Th i s system can sometimes be s t a ined wi th 6-pgdh - on same g e l by adding 6-phosphogluconfc ac id t o above.

TABLE 3. Continued.

P ro t e in Rat ing Technique

GOT 1 To 50 m l of Trizma 8.0 ( s o l u t i o n 13) add: 1 m l L-Aspartic Acid ( s o l u t i o n 11) 1 m l LA. -Ketoglu tar ic Acid ( s o l u t i o n 12) 10 mg Pyridoxal-5 '-Phosphate Pcur over g e l and incubate f o r 30 minutes a t 3 7 O ~ . Add 50 mg F a s t Blue BB S a l t and incubate f o r 2 hours more. Remove s t a i n and add wash ( s o l u t i o n 18).

Hk* 1

To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 80 mg Na4Fructose-1,6-diphosphate*6H20 35 u n i t s Aldolase 1 m l MTT ( s o l u t i o n 20) 2 m l DPN ( s o l u t i o n 22) 100 mg Sodium Ar sena te . Pour over g e l and incubate f o r 20 minutes a t 37 '~. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate 2 hours more. Remove s t a i n and add wash ( s o l u t i o n 18).

To 35 m l Trizma 7.2 ( s o l u t i o n 14) add: 50 mg d - g l y c e r o p h o s p h a t e 1 m l MTT ( s o l u t i o n 20) 2 m l DPN ( s o l u t i o n 22).

0 Pour over g e l and incubate f o r 30 minutes a t 37 C. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate f o r 1 hour more. Remove s t a i n and add wash ( s o l u t i o n 18). NOTE: This system may be s t a i n e d simultaneously w i t h - Adh by adding 2 m l I sopropyl Alcohol t o above.

To 35 m l of Trizma 7,2 ( s o l u t i o n 14) add: 50 mg 4-D+-Glucose 1 m l MgC12 ( s o l u t i o n 16) 1 m l MTT ( s o l u t i o n 20) 1 m l TPN ( s o l u t i o n 21) 2 m l G-6-pdh ( s o l u t i o n 23) 40 mg ATP. Pour over g e l and incubatz a t 3 7 O ~ f o r 30 minutes. Add 0.5 m l of PMS ( s o l u t i o n 19) and incubate f o r 1 hour more. Pour o f f s t a i n , f i l t e r , and save f o r reuse. Add wash ( s o l u t i o n 18). NOTE: Idh sometimes shows up on t h i s system. Addi t ion - of 15 mg of DL-Na3-Isocitric Acid e s p e c i a l l y enhances t h i s e f f e c t . This s t a i n must have good q u a l i t y G-6-pdh.

TABLE 3. Continued.

P ro t e in Rat ing Technique

Id h* 1 To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 50 mg DL-Na3-Isocitric Acid 4 m l MnC12 ( s o l u t i o n 17) 1 m l MTT ( s o l u t i o n 20) 1 m l TPN ( s o l u t i o n 21) . Pour over g e l and incubate a t 3 7 ' ~ f o r 30 minutes. Add 0.5 m l of PMS ( s o l u t i o n 19) and incubate f o r 1 hour more. Remove s t a i n and add wash ( so lu t ion 18). NOTE: This system can a l s o be s t a ined on Hk; s e e above. -

1 To 50 m l of Maleate b u f f e r ( s o l u t i o n 3) add: 1 m l L-leucyl- .!4 -napthylamide ( s o l u t i o n 1) . Pour over g e l and incubate a t room temperature f o r 15 minutes. Add 3 / 4 m l of Black K s a l t ( s o l u t i o n 2). Incubate a t 37OC f o r 2 hours. Remove s t a i n and add wash ( s o l u t i o n 18) .

LAP

ME and IYIdh* 1 To 35 r n l 02 Trizma, 7 - 2 ( s o l u t i o n 14) add: 1 m l MTT ( s o l u t i o n 20) 1 m l TPN ( s o l u t i o n 21) 1 m l DL-malate ( s o l u t i o n 24) . Pour over g e l and incubate a t 37O f o r 30 minutes. Add 0.5 m l PMS ( s o l u t i o n 19). When ME appears , add 2 m l DPN ( s o l u t i o n 22) f o r Mdh. Incubate 30 minutes more, d r a i n o f f s t a i n , f i l t e r and save f o r reuse. Add wash ( s o l u t i o n 18).

Odh* 2 Disso lve 2 m l Octanol i n 2 m l Ethanol:Methanof (1:l) mixture. Add t h i s t o : 35 m l Trizma 8 ,8 ( s o l u t i o n 13) 1 m l MTT ( s o l u t i o n 20) 2 m l DPN ( s o l u t i o n 22)" Incubate a t 37OC for 45 minutes. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate f o r 2 hours more. Pour o f f s t a i n , f i l t e r and save f o r reuse. Add wash ( s o l u t i o n 18). NOTE: Adh w i l l appear on g e l a l s o due t o presence of - Ethanol. Xdh may be s t a i n e d f o r by d i s so lv ing Xanthine i n Ethanol a l s o .

3 To 35 m l Trizma 7.2 ( s o l u t i o n 14) add: 4 m l MgCl ( s o l u t i o n 16) 1 m l MTT ? s o l u t i o n 20) % m l TPN ( s o l u t i o n 21) 1 m l G-6-pdh ( s o l u t i o n 23) 50 mg Fructose-6-phosphate. Incubate f o r 30 minutes a t 37OC. Add 0.5 m l PMS ( so lu t ion 19) and incubate f o r 1 hour more. Pour o f f s t a i n and add wash ( s o l u t i o n 18).

TABLE 3. Concluded.

P ro t e in Rat ing Technique

Pgm* 1 To 35 m l Trizma 7.2 ( s o l u t i o n 14) add: 4 m l MgC12 ( s o l u t i o n 16) 1 m l MTT ( s o l u t i o n 20) 1 m l TPN ( s o l u t i o n 21) 4 m l G-6-pdh ( s o l u t i o n 23) 4 m l Glucose-1,6-diphosphate ( s o l u t i o n 25) 4 m l Glucose-1-phosphate ( s o l u t i o n 26). Pour over g e l and incubate f o r 30 minutes a t 370C. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate f o r 1 hour more. Remove s t a i n , f i l t e r and save f o r reuse. Add wash ( s o l u t i o n 18). NOTE: This s t a i n must have good q u a l i t y G-6-pdh t o work. -

6-pgdh* 3 To 35 m l Trizma 7.2 ( s o l u t i o n 14) add: 50 mg 6-phosphogluconic Acid 1 m l MTT ( s o l u t i o n 20) .

1 m l TPN ( s o l u t i o n 21). Pour over g e l and incubate a t 3 7 ' ~ f o r 30 minutes. Add 0.5 m l PMS ( s o l u t i o n 19) and incubate f o r 1 hour more. Remove s t a i n and add wash ( s o l u t i o n 18). NOTE: This s t a i n can sometimes be s t a ined wi th G-6-pdh - on t h e same g e l by adding Glucose-6-Phosphate t o above.

Xdh* 2 This s t a i n w i l l not work un le s s t he s u b s t r a t e , Hypoxanthine, i s d isso lved . Th i s may be accomplished by:

(1) Boi l ing 100 mg i n 50 m l Trizma 7.2 ( s o l u t i o n 14); (2) Disso lv ing 100 mg i n 10 m l .2 N HC1 and, a£ t e r

d i s s o l u t i o n , br inging t h e pH back t o 7.0-8.0 wi th Trizma base; o r

(3) Disso lv ing 100 mg i n 5 m l of Ethanol o r Methanol.

To 35 m l of Trizma 7.2 ( s o l u t i o n 14) add: 100 mg Hypoxanthine d isso lved i n 5 m l Ethanol 1 m l MTT ( s o l u t i o n 20) 2 m l DPN ( s o l u t i o n 22). Pour over g e l and incubate a t 37OC f o r 40 minutes. Add 0.5 m l of PMS ( s o l u t i o n 19) and incubate f o r 2 hours more. Remove s t a i n , f i l t e r , and save f o r reuse. Add wash t o g e l ( s o l u t i o n 18). NOTE: Adh w i l l appear on g e l a l s o due t o presence of - Ethanol. Xdh may a l s o be s t a ined wi th Odh.

Rat ings: 1 I n r e g u l a r use i n our lab , d i s p l a y s good banding p a t t e r n s , found i n a l l Hawaiian Drosophila i nves t iga t ed t o da t e .

2 Usual ly d i sp l ays good banding p a t t e r n s , not found i n a l l spec i e s of Hawaiian Drosophila i nves t iga t ed t o da te .

3 Works spo rad ica l ly w i th poor band resolvement f o r most Hawaiian Drosophila spec i e s ; r e q u i r e s development of a d d i t i o n a l ge l - e l ec t rode b u f f e r systems.

A f t e r t h i s s t e p , about 50-75 m l of wash s o l u t i o n (TABLE 4, s o l u t i o n 18)

should be poured upon t h e g e l s l i c e . This w i l l s e rve t h e purpose of c l e a r i n g

background s t a i n from t h e g e l s l i c e and a c t t o preserve it. However, i t i s a

good idea t o record ( score) t h e d a t a be fo re adding the wash s o l u t i o n , a s some

s t a i n s w i l l f ade i n wash w i t h i n s e v e r a l hours. A l l TPN and DPN dependent enzymes

w i l l f ade a t room temperature over s e v e r a l months. It is wise t o photograph o r

make a drawing of t h e g e l s l i c e a s soon a s poss ib le . I f it i s des i r ed t o save

these , i t is b e s t t o wrap them i n sa ran wrap and s t o r e them a t a very c o o l

temperature a f t e r soaking i n wash s o l u t i o n ~ o v e r n i g h t .

GEL AND ELECTRODE BUFFER SYSTEMS

We have found four bu f fe r systems u s e f u l i n a n a l y s i s of Hawaiian Drosophil id

p r o t e i n systems (TABLE 1). The f i r s t two a r e modified from Poulik (1957) and

a r e d iscont inuous i n na ture ; t h a t i s , t h e br idge (e lec t rode) b u f f e r s a r e

d i f f e r e n t from t h e g e l bu f fe r s . The t h i r d system (System C) i s a cont inuous

system wi th the b r idge bu f fe r having t h e same composition a s t he g e l b u f f e r , but

being much more concentrated. This system i s modified from Shaw and Prasad

(1970) and has t h e unique f e a t u r e of having a h igher i on ic concen t r a t ion i n t he

ca thodal t r a y than i n t h e anodal b u f f e r chamber. F i n a l l y , t h e f o u r t h system i s

a l s o a d iscont inuous system and i s modified a f t e r Ashton and Braden (1961).

TABLE 2 l i s ts the enzyme o r p r o t e i n systems which can be analyzed on each

bu f fe r system. Note t h a t some systems can be analyzed on the same g e l ( i . e . Xdh,

Odh and Adh on System A ; Mdh and ME on System C and &\-gpdh and Adh on System

C). Since a maximum of 5 g e l s l i c e s can be obtained without much d i f f i c u l t y from

each 1 cm t h i c k g e l , t h i s "double-s taining" technique enables one t o s t a i n f o r

5-8 p r o t e i n systems on each 1 cm t h i c k g e l , Some of t hese systems i n t u r n code

TABLE 4. Stock s o l u t i o n s . A l l wate r used should be deionized and d i s t i l l e d .

So lu t ion Number Name To Make To Use

1 L-leucyl- 6 -napthylamide 1 gr.140 m l H20 (LAP s u b s t r a t e )

1 m1/50 s o l u t i o n 3

2 Black K S a l t (LAP s t a i n )

1 gr , /25 m l H20 314 m1/50 m l s o l u t i o n 3

3 Maleate b u f f e r (LAP s t a i n bu f f e r )

24.2 gr . Trizma Base (Sigma) 50 m l , use w i th s o l u t i o n s 23.2 gr . Maleic Acid 1 and 2. 1000 m l H20; pH'5.2

4 b - n a p t h y l Ace t a t e 1 gr.150 m l Acetone (Es s u b s t r a t e s r e d bands on g e l )

--

1 m1/50 m l ( see s o l u t i o n 7)

5 .d, -napthy1 Ace ta t e 1 gr./25 m l Acetone 4- 25 m l H20 1 m1/50 m l ( s ee s o l u t i o n 7) I

(Es subs t r a t e=b lack bands) - +' Cn

6 NaP04A 0.2M Plonobasic Sodium Phosphate Mix 50 m l Nap04 A w i t h 10 t

(Es s t a i n bu f f e r , a c i d i c pH) (13.2 gr.1500 m l H20) a1 Nap04 B ( s o l u t i o n 7 ) , t hen S t o r e i n da rk b o t t l e add 40 m l Hz0 (pH=6.1)

7 NaP04B 0.2 M Dibas ic Sodium Phosphate Mix 10 m l t o 50 m l s o l u t i o n 6, (Es s t a i n bu f f e r , b a s i c pH) (26.81 gr.1500 m l H20) add 40 m l Hz0 (pH=6.1),

S t o r e i n da rk b o t t l e add s u b s t r a t e s t o t h i s s o l u t i o n

8 Ace ta t e A 23.1 m l G l a c i a l Ace t i c A c i d 1 2 ~ H20 Mix 15 m l Ace t a t e A w i t h 35 (Acph s t a i n b u f f e r , a c i d i c pH) m l s o l u t i o n 9 , add 50 m l H20

9 Ace ta t e B 27.2 gr . Sodium ace tat ell^ H20 (See s o l u t i o n 8 ) (Acph s t a i n b u f f e r , b a s i c pH)

10 Borate 5.0 (Acph presoak bu f f e r )

-- --- -

30.9 g r , Boric AcidI lL H20 (0.5M) Pour 50 m l on g e l f o r % hour, Bring t o pH=5.0 w i t h 1 M Trizma Base pour o f f , add Acph s t a i n

11 L-aspa r t i c Acid (GOT s u b s t r a t e )

100 mg/ml H20 Add 1 m l t o 50 m l s o l u t i o n 12

TABLE 4. Continued.

-- --

So lu t ion Number . Name To Make To Use

12 Cr6 - k e t o g l u t a r i c Acid (GOT s u b s t r a t e )

75 mglml H20 add 1 m l t o 50 m l s o l u t i o n 12

13 Trizma b u f f e r 8.0 24.2 g r . Trizma Base (0.2M) Mix 50 m l w i th s o l u t i o n s (GOT and Odh s t a i n bu f f e r ) 11.8 m l concent ra ted HC1 11 and 12, pH=8.0

1000 m l H20

14 Trizma b u f f e r 7.2 (genera l s t a i n bu f f e r )

12.1 g r . Trizma Base (0.1M) 7.5 m l concent ra ted HC1

-- ---

Mix 35 m l w i th TPN and DPN dependent s t a i n s , pH37.2 ( s ee TABLE 3)

15 Trizma b u f f e r 10.6 24.2 g r . Trizma Base ( 2 ~ ) / 1 ~ H20 pour 50 m l on g e l f o r % hour (Ap presoak bu f f e r ) pH=10.6 pour o f f , t hen add s t a i n f o r Ap -

16 MgC12 2.03 g r . (0.1M)/100 m l H20 See s t a i n s , TABLE 3 -

17 MnC12 4.9 g r , ( 0 . 2 5 ~ ) / l o 0 m l H20 See s t a i n s , TABLE 3

18 Wash s o l u t i o n 5 p a r t s Ethanol o r Methanol pour 50 m l on g e l a f t e r 5 p a r t s Hz0 s t a i n i n g ; l e ave on overn ight 1 p a r t G l a c i a l Ace t i c Acid

19 Phenazine Methosulfate ( PMS

0.5 m1/35 m l s t a i n ( see TABLE 3)

20 WT Tetrazol ium 1 gr.1100 m l H20 1 m1/35 m l s t a i n ( s t a i n f o r TPN and DPN dependent s t a i n s ) ( see TABLE 3)

21 Triphosphopyridine Nucleot ide 1 gr./lOO m l H20 1 m1/35 m l s t a i n ( T W ( see TABLE 3)

22 Diphosphopyridine Nuc l e o t i d e 1 gr.1100 m l H20 2 m1/35 m l s t a i n ( D m ( see TABLE 3)

TABLE 4. Concluded.

S o l u t i o n Number Name To Make To Use

23 Glucose-6-phosphate dehydrogenase (G-6-pdh)

Use 3 m1/35 m l s t a i n ( s ee TABLE 3)

--

24 DL-Malate 268.2 g r . ( ~ . O M ) / ~ L H20 Use 1 m1/35 m l s t a i n ( s u b s t r a t e f o r ME and Mdh) Adjus t t o pHz7.0 w i th 150 gr . NaOH

25 c4,-D-~lucose- 1 ,6-diphosphate 22 mg (0 .0004~) / I00 m l H20 Add 3 m1/35 m l s o l u t i o n 14 (Pgm s u b s t r a t e ) \

-- - -- -

2 6 d-D-Glucose- l -phosphate 1.7 gr . (0 .046~) /100 m l H20 Add 3 m1/35 m l s o l u t i o n 14 (Pgm s u b s t r a t e )

27 Coomassie B r i l l i a n t Blue 1 g r . / l ~ s o l u t i o n 18 CBB (General P r o t e i n s t a i n )

Pour 50 m l over g e l (save f o r reuse)

28 Ammonium P e r s u l f a t e A PS

10 gr./100 m l H20 (10% s o l u t i o n ) Use 0.4 m1/100 m l g e l b u f f e r t o make Acrylamide g e l s ( see s e c t i o n V , t e x t )

f o r more than one locus. The technique opt imizes , then, t h e amount of informa-

t i o n which can be gained from a s i n g l e smal l organism such a s a f l y .

THE USE OF ACRYMMI.DE GELS

Acrylamide has proven t o be extremely u s e f u l i n e l ec t rophores i s . It o f f e r s

a b e t t e r method t o c o n t r o l pore s i z e of t h e g e l , leading t o c l e a r e r and more

def ined banding p a t t e r n s i n some in s t ances . Being t r anspa ren t , i t f a c i l i t a t e s

q u a n t i f i c a t i o n of p ro t e ins by densiornetric methods. I n add i t i on , s t a r c h sometimes

has i ons p re sen t which w i l l i n t e r f e r e w i t h some p r o t e i n assays. Acrylamide i s

a l s o e a s i e r t o handle ( i . e . t h e g e l can be prepared and ready f o r use i n 20

minutes a s i t r e q u i r e s no cooking), and it runs e l e c t r o p h o r e t i c a l l y f a s t e r than

s t a r c h .

Acrylamide does have s e v e r a l i nhe ren t problems. It i s q u i t e t o x i c , and

c a r e should be taken t o avoid con tac t w i th ba re s k i n and t o prevent i n h a l a t i o n .

Because of i t s h igh t e n s i l e s t r e n g t h , acrylamide is extremely hard t o c u t i n a

h o r i z o n t a l plane. For t h i s reason i t i s d i f f i c u l t t o use i n popula t ion surveys

where more than t h r e e p r o t e i n systems a r e under a n a l y s i s , a l though i t w i l l work

f i n e where t ime, expense and sample s i z e s a r e not i n s h o r t supply.

I n genera l , those p r o t e i n systems a s sayab le on s t a r c h a l s o work on

acrylamide. However, c a r e should be taken t o keep t h e amount of ammonium

p e r s u l f a t e (APS) t o a minimum a s t h i s w i l l i n h i b i t p r o t e i n a c t i v i t y a t medium

concent ra t ions . To prevent APS i n h i b i t i m , i t i s sometimes wise t o f i r s t run a

c u r r e n t of 50-60 mA through t h e g e l f o r 3 hours before samples a r e placed i n the

g e l . The technique serves t o c l e a r t h e g e l of i n h i b i t o r y f a c t o r s .

We have found t h a t a shal low g e l mold ( i . e . a th inner g e l s l a b ) y i e l d s

b e t t e r def ined p r o t e i n systems. For t h i s reason, ve rou t ine ly use s l a b molds

about 19.5 cm x 17.5 cm x 2 mm (volume = 100 ml) i n s i z e .

Note t h e amount of acrylamide (Cyanogum 41, F i s h e r S c i e n t i f i c , F a i r Lawn, s i z e

N. J.) w i l l vary i n concen t r a t ion depending on the g e l pore l requi red f o r proper

s e p a r a t i o n of a i lozymic p ro t e ins . General ly , t h e l a r g e r t h e p r o t e i n molecule,

t h e l a r g e r t he pore s i z e should be t o prevent molecular f r a c t i o n a t i o n .

The same g e l and e l ec t rode b u f f e r s used i n s t a r c h g e l s can be u t i l i z e d f o r

acrylamide ge l s . The fol lowing method has been adapted f o r use i n t h i s l a b and

has been modified from Raymond and Weintraub (1959):

To LOO m l g e l bu f fe r add, i n o rde r ;

6-11 grams Cyanogum 41, d i s so lve ,

0.1 m l of TEMED (N,N,N',N'-tetramethylethylenediamine, Eastman Kodak,

Rochester, N. Y .) , 0.4 m l 10% APS

Qu ick ly mix and pour i n t o g e l mold. Cover mold wi th g l a s s p l a t e

(19.5 x 18 cm), tak ing c a r e t h a t no a i r bubbles occur a t t h e i n t e r f a c e of t h e

g l a s s and t h e g e l l i n g so lu t ion . Let s e t f o r 20-30 minutes, then g e n t l y remove

the g l a s s p l a t e . Use g e l a s d i r e c t e d i n s e c t i o n 111 and above.

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TECHNICAL REPORTS OF THE US/IBP ISLAND ECOSYSTEMS IRP

No. 1

No. 2

No, 3

No. 4

No. 5

No. 6

No, 7

No. 8

No. 9

No* 10

No. 11

No. 12

No. 13

No. 14

No. 15

( In t eg ra t ed Research Program)

Hawaii T e r r e s t r i a l Biology Subprogram, F i r s t Progress Report and Second- Year Budget. D. Mueller-Dombois, ed. December 1970. 144 p.

I s l a n d Ecosystems S t a b i l i t y and Evolu t ion Subprogram. Second Progress Report and Third-Year Budget. D. Mueller-Dombois, ed. January 1972. 290 p.

The in f luence of f e r a l goa t s on koa (Acacia koa Gray) reproduct ion i n Hawaii Volcanoes Nat iona l Park. G. Spatz and D. Mueller-Dombois, February 1972. 16 p.

A non-adapted vege t a t i on i n t e r f e r e s w i th s o i l water removal i n a t r o p i c a l r a i n f o r e s t a r e a i n Hawaii. D. Mueller-Dombois. March 1972. 25 p.

Seasonal occurrence and h o s t - l i s t s of Hawaiian Cerambycidae. J. L. Gressitt and C. J. Davis. A p r i l 1972. 34 p.

Seed d i s p e r s a l methods i n Hawaiian Metrosideros. Carolyn Corn. August 1972. 19 p.

Ecologica l s t u d i e s of C tenosc i a r a hawai iens i s (Hardy) (Diptera: Sc i a r idae ) . W . A. S t e f f a n . August 1972. 7 p.

Bi rds of Hawaii Volcanoes Na t iona l Park. A. J. Berger. August 1972. 49 p.

B ioene rge t i c s of Hawaiian honeycreepers: t h e Amakihi (Loxops virens) and t h e Anianiau (L. ~ a r v a ) . R. E. MacMillen. August 1972. 14 p.

Invas ion and recovery of v e g e t a t i o n a f t e r a vo l can i c e r u p t i o n i n Hawaii, G, A, Smathers and D. Mueller-Dombois, September 1972. 172 p.

Bi rds i n t h e Kilauea F o r e s t Reserve, a p rogress r e p o r t . A. J. Berger. September 1972. 22 p.

E ~ o g e o g r a ~ h i c a l v a r i a t i o n s of chromosomal polymorphism i n Hawaiian popula t ions of Drosophi la immiarans. Y. K, Paik and K. C. Sung. February 1973. 25 p.

The in f luence of f e r a l goa t s on t h e lowland vege t a t i on i n Hawaii Volcanoes Na t iona l Park. D. Mueller-Dombois and G. Spatz . October 1972. 46 p.

The in f luence of SO2 fuming on t h e v e g e t a t i o n surrounding t h e Kahe Power P l a n t on Oahu, Hawaii. D. Mueller-Dombois and G. Spatz . October 1972. 12 p.

Succession p a t t e r n s a f t e r p i g d igg ing i n grass land communities on Mauna Loa, Hawaii, G. Spatz and D. Mueller-Dombois. November 1972. 44 p.

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Ecological s tud ies on Hawaiian lava tubes. F. G. Howarth. December 1972. 20 p.

Some f indings on vegeta t ive and sexual reproduction of koa. GUnter 0. Spatz. February 1973. 45 p.

A l t i t u d i n a l ecotypes i n Hawaiian Metrosideros. Carolyn Corn and William Hiesey. February 1973. 19 p.

Some aspects of is land ecosystems analys is . Die ter Mueller-Dombois. February 1973. 26 p.

F l i g h t l e s s Dolichopodidae (Diptera) i n Hawaii. D. Elmo Hardy and Mercedes D. Delfinado. February 1973. 8 p.

Third Progress Report and Budget Proposal f o r FY 74 and FY 75. D. Mueller- Dombois and K. Bridges, eds. March 1973. 153 p.

Supplement 1. The cl imate of the IBP s i t e s on Mauna Loa, Hawaii. Kent W. Bridges and G. Virg in ia Carey. Apr i l 1973. 141 p.

The bioecology of Psvl la uncatoides i n the Hawaii Volcanoes National Park and the Acacia koaia Sanctuary. John R. Leeper and J. W. Beardsley. A p r i l 1973, 13 p.

Phenology and growth of Hawaiian p lants , a preliminary repor t . Charles H. Lamoureux. June 1973. 62 p.

Laboratory s tud ies of Hawaiian Sciar idae (Diptera). Wallace A. S tef fan . June 1973. 17 p.

Natural a rea system development f o r the Pac i f i c region, a concept and symposium. Die ter Mueller-Dombois. June 1973. 55 p.

The growth and phenology of Metrosideros i n Hawaii. John R. Porter . August 1973. 62 p.

EZPLOT: A computer program which allows easy use of a l i n e p l o t t e r , Kent W. Bridges, August 1973. 39 p.

A reproductive biology and n a t u r a l h i s to ry of the Japanese white-eye (Zosterops J.a.onica iaponica) i n urban Oahu. Sandra J. Guest. September 1973. 95 p. '

Techniques f o r e lec t rophores is of Hawaiian Drosophila. W. W . M. S t e i n e r and W . E, Johnson. November 1973. 21 p.