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A simple apparatus for retrieving density gradients by surface tension
A Simple
Apparatus
for Retrieving by Surface Tension
Density
Gradients
90
polyethylene tubing: was clrill~cl itt tltcb ccntc~r of tltc disc. The twl of tltc polyet,hylcne tubing neat-m tltc coil was lmss~~l through this ltolc and flared slightly 1)~ ltol~ling tltc> md ttcar :L flnntc~. T11c fl:tre keeps the disc cottttcctcd to llic ttihing n-ltilo still allowittg it to gimbal freely. This allows tltc disc to rcnmitt p~rpetttliculur to the long axis of the centrifuge tube rcgardlese of the angle assurnwl hp tltc ctt(l of the J)olyetltylcnc itt relation to tlicl long axis of the cetttrifugc~ tube. To opcratc tltc device, the following rcgitnen was uwtl. (1 1 Tltc tol) of the coil was taped to :I supporting rod, :tu(l tltc plastic disc was wtttcwtl 2 cm above tltc top of the grntlicnt. (2) Tlt(, clisc was carefully J~twrctl toward the grnclimt surfaw tuttil it be~xtnc attncltvtl I)y surfaw tvtision. (.?I A syringe v-:1!: usc~l to start the siphott ant1 rcmo~~~l wltm t1ti.s wts :~c~omJ~lishc~d. Tltv flow rate co17111tlivti 1~ :Kljlistc(l to tltc tlcsircvl r:ttc by mryitig that llcigltt of tltv frw ctt(l of tl!cl ~~olycthyl~tic ttthing. Ihtrittg oJwrstion, tlto dcsccnditig fluid surface pttllr~l the plastic disc with it, ant1 tltc polycth;r-lrne coil strctchcd to provide thr additional lengt,lt. The dmiw vxs tcstcvl on n continnolts grndicmt forrnctl })p :I Ruclllrt* linear gr:tdientm tnnkm- iBuchlrr Itwtrurncnt:: J~ivision, N.J) in a 28.6 x 103.5 mui round bottom polpm-honntt~ wntrifugc tuhc. The grntlicnt was fortned from 19 ml of R 2056 (q/k) solution of sucrose and 19 1111 of a 107% (wj’w) solution of sucrose. Ucforc th(> grnrlic~nt ~~2sadrlfyl to the cent,rifug;c tube, ii ml of tltv 2Ocljcsucrose iv:ts :~dclwl t3 fill tltc round
FRACTION
FI(;. 2. .I plot of thr ha11 a I-olume 0.
Points
of 2 ml.
wsociated
NUMBER
omolarity versus fraction number is shown. Each fraction Points associntrd mith line A are represented by t,he symbol with line R are v~prrrcntrd 1)~ t,hc syml)nl A.
92
GLEN
S. GERMAIN
regimen previously described. Again, 2-ml aliquots were collected, the osmolarities determined. Line A in Fig. 2 is the least-square determined by these points.
and line
CONCLUSIONS
A linear gradient retrieved by the described apparatus follows closely the gradient as it is present in the centrifuge tube before removal. The small discrepancy between thcb two lines can be explained by variations in the gradient produced by the out,put as it runs down the side of the centrifuge tube, causing some mixing of the various levels. This problem, together with errors in measuring the osmolarity and those introduced by the retrieval system, would account for the observed differences. The question of which factor causes the greatest deviation from linearit,y is difficult to answw. Thcl fractions roll&cd directly from the gradient marker did not fall exactly on a straight line, indicating t.hat one or both of the measurements of osmolarity and the shape of the gradient produced introduce some error. Also, it is arsumcd that the gradient maker produces a linear gradient every time. This can only bc verified in this system by retrieving the gradient, ther&F making it, difficult to differentiate between nonlinearity in the original gradient and that introduced by the retrieval syst,em. The ease of fabricat’ion and operation of this device, together with its accuracy and negligible costs, recommend it as a useful tool. ACKNOWLEDGMENT8 T.
Thr support, of Dr. E. E. Muirhrad Kirk in the preparation of graph
and t,he nssistmw of nlr. 77’. Brosius 2nd photograph is appreciated.
anti
REFERENCES 1. BOONE. C. Iv., HARP:I.L, G. $., AXI) BOND. II. 2. Fox. T. 0.. ANI) PARIM, iz. 13. (19io) tkimrf 3. LEIF, R. C. (1968) i2nnl. Bioch~m. 25, 271.
E. IfiT,
(1968) 80.
J. Cell
Rid.
36, 369
Mr.
Apparatus
for Retrieving by Surface Tension
Density
Gradients
90
polyethylene tubing: was clrill~cl itt tltcb ccntc~r of tltc disc. The twl of tltc polyet,hylcne tubing neat-m tltc coil was lmss~~l through this ltolc and flared slightly 1)~ ltol~ling tltc> md ttcar :L flnntc~. T11c fl:tre keeps the disc cottttcctcd to llic ttihing n-ltilo still allowittg it to gimbal freely. This allows tltc disc to rcnmitt p~rpetttliculur to the long axis of the centrifuge tube rcgardlese of the angle assurnwl hp tltc ctt(l of the J)olyetltylcnc itt relation to tlicl long axis of the cetttrifugc~ tube. To opcratc tltc device, the following rcgitnen was uwtl. (1 1 Tltc tol) of the coil was taped to :I supporting rod, :tu(l tltc plastic disc was wtttcwtl 2 cm above tltc top of the grntlicnt. (2) Tlt(, clisc was carefully J~twrctl toward the grnclimt surfaw tuttil it be~xtnc attncltvtl I)y surfaw tvtision. (.?I A syringe v-:1!: usc~l to start the siphott ant1 rcmo~~~l wltm t1ti.s wts :~c~omJ~lishc~d. Tltv flow rate co17111tlivti 1~ :Kljlistc(l to tltc tlcsircvl r:ttc by mryitig that llcigltt of tltv frw ctt(l of tl!cl ~~olycthyl~tic ttthing. Ihtrittg oJwrstion, tlto dcsccnditig fluid surface pttllr~l the plastic disc with it, ant1 tltc polycth;r-lrne coil strctchcd to provide thr additional lengt,lt. The dmiw vxs tcstcvl on n continnolts grndicmt forrnctl })p :I Ruclllrt* linear gr:tdientm tnnkm- iBuchlrr Itwtrurncnt:: J~ivision, N.J) in a 28.6 x 103.5 mui round bottom polpm-honntt~ wntrifugc tuhc. The grntlicnt was fortned from 19 ml of R 2056 (q/k) solution of sucrose and 19 1111 of a 107% (wj’w) solution of sucrose. Ucforc th(> grnrlic~nt ~~2sadrlfyl to the cent,rifug;c tube, ii ml of tltv 2Ocljcsucrose iv:ts :~dclwl t3 fill tltc round
FRACTION
FI(;. 2. .I plot of thr ha11 a I-olume 0.
Points
of 2 ml.
wsociated
NUMBER
omolarity versus fraction number is shown. Each fraction Points associntrd mith line A are represented by t,he symbol with line R are v~prrrcntrd 1)~ t,hc syml)nl A.
92
GLEN
S. GERMAIN
regimen previously described. Again, 2-ml aliquots were collected, the osmolarities determined. Line A in Fig. 2 is the least-square determined by these points.
and line
CONCLUSIONS
A linear gradient retrieved by the described apparatus follows closely the gradient as it is present in the centrifuge tube before removal. The small discrepancy between thcb two lines can be explained by variations in the gradient produced by the out,put as it runs down the side of the centrifuge tube, causing some mixing of the various levels. This problem, together with errors in measuring the osmolarity and those introduced by the retrieval system, would account for the observed differences. The question of which factor causes the greatest deviation from linearit,y is difficult to answw. Thcl fractions roll&cd directly from the gradient marker did not fall exactly on a straight line, indicating t.hat one or both of the measurements of osmolarity and the shape of the gradient produced introduce some error. Also, it is arsumcd that the gradient maker produces a linear gradient every time. This can only bc verified in this system by retrieving the gradient, ther&F making it, difficult to differentiate between nonlinearity in the original gradient and that introduced by the retrieval syst,em. The ease of fabricat’ion and operation of this device, together with its accuracy and negligible costs, recommend it as a useful tool. ACKNOWLEDGMENT8 T.
Thr support, of Dr. E. E. Muirhrad Kirk in the preparation of graph
and t,he nssistmw of nlr. 77’. Brosius 2nd photograph is appreciated.
anti
REFERENCES 1. BOONE. C. Iv., HARP:I.L, G. $., AXI) BOND. II. 2. Fox. T. 0.. ANI) PARIM, iz. 13. (19io) tkimrf 3. LEIF, R. C. (1968) i2nnl. Bioch~m. 25, 271.
E. IfiT,
(1968) 80.
J. Cell
Rid.
36, 369
Mr.