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A simple and inexpensive method for the concentration of protein solutions by means of ultrafiltration
CLINICA
CHIMICA
375
ACTA
A simple and inexpensive method fur the concentration of protein solutions by means of ultrafiltration* We have tested a simple method for the concentration avoids (I) transfer of the material to be concentrated,
of protein solutions which loss of proteins on the
(2)
membrane of an ultr~~filtration apparatus after complete passage of the liquid due to inaccurate timing, (3) loss of proteins caused by denaturation through drying (in evaporation and carbowax methods), and (4) the passage of smal1 molecules into the protein solution from externa1 hypertonic solutions (carbowax method) which may disturb c~~somatagra~hy and/or fingel-printing if enzymatic degradation studies of the protein have to be done. d
a
b
aa
c
e
c
a
glass
plate
b
flier
paper
c
microscoDe
e
dialysis
slide bag
The method requires two glass plates and weights of approximately IO-IS kg. The protein solution to be concentrated is put into a dialysis bag (Visking type, measuring B/32 in diameter was used). This is done in such a way that no spare room is left in the bag. Each end has two sturdy knots. The dialysis bag is then placed on a sheet of filter paper, which in its turn lies on one of the glass plates, Next to the dialysis hag, 4 microscope slides (0.8 mm, thick) are placed, z on each side. Another sheet of filter paper covers the bag and slides. The second glass plate goes on top of it and the weights are placed on top (Fig. I). A significant loss of fluid from the bag will be observed by 4 h. The rate of ultra~ltr~tion is maintained by keeping the bag tense. This is easily accomplished by twisting the bag, beginning at one end. The twisting wiil squeeze the remaining Auid into a single section. A knot is tied in the twisted, empty portion and the now tense bag is placed under pressure again. The volume can easily be reduced to one-tenth of the original in a4 h. The method is preferably performed in a cold room or refrigerator. Dialysis, if needed, can be done before or after concentration of the protein solution. One or more bags can be “squeezed” simultaneously. In the last case the rate of concentration is the same for each of them and is theoretically independent of their relative volumes. * The wurk was supported HE-04706 and WB 04038.
in part by grants from the United States Public Health Service;
CEB?l. Chitn. Acra, 10 (rc364)37j-376
376
SHORT
COMMUNICATIONS
The above mentioned microscope slides are to prevent complete loss of liquid from the dialysis bag. To serve a specific demand, even thinner or also thicker layers may be used. Quantitation of the rate of concentration can be estimated (a) roughly, by measuring and comparing the length of the dialysis bag before and after ultrafiltration (each time in the state of minimum bag volume), (b) more accurately, by weighing the bag before and after concentration (mind wet weight of the bag at the start) or (c) by comparing the O.D.‘s before and at the end of the procedure. Central Laboratory of the Netherlands Red Cross Blood Transfusion Service,
F. PEETOOM PARK
S. GERALD
Amsterdam
(The Netherlands), and Clinical Genetics Laboratory, Children’s Hospital Medical Center, Boston 15, Mass. (U.S.A). Received
April zrth,
1964 Clin.Chinz.
An improved method for calcium determination
Acta, IO (1964)375-376
in serum
The determination of serum calcium according to the FERRO AND HAM method’ using chloranilic acid, is very convenient; however it requires rather large volumes of serum. The method was subsequently modified by the same authors2 by increasing the concentration of chloranilic acid and using microcuvettes for spectrophotometric readings, thus allowing determinations with 0.5 ml of serum. An increase in sensitivity was obtained also by the addition of a 1% FeCl, solution freshly prepared each times. The observation that a sharp increase in optical density of chloranilic acid solutions is obtained at pH 1.5-1.6 (Fig. I), made it possible to increase about fourfold the sensitivity of the method (Fig. z), and thus to operate with 0.5 ml of serum without the need of special apparatuses and using only stable reagents. Reagents (I)
Chloranilic acid solution: I g of chloranilic acid (BDH) is dissolved in 80 ml of water by addition of 7 ml of I N NaOH; the pH is adjusted in a range between 3.5 and 7 by further addition of NaOH or chloranilic acid. The volume is then taken to IOO ml. After 24 h in refrigerator at 4”, the solution is filtered through Whatman No. I filter paper and stored in a brown bottle. Eventual further precipitates
are discarded
by filtration
before use.
(2) Isopropyl alcohol-water 50 : 50 (v/ v). (3) 5% solution of tetrasodium salt of ethylendiaminetetraacetic
acid (BDH).
(4) 0.1 M glycine buffer at PH 1.5. (5) Standard solution of Ca++: 0.2497 g of CaCO, are dissolved in IO ml of I N HCl and taken to 1000 ml with distilled water. A concentration of IO mg Ca++ per IOO ml is thus obtained. Clin. Chim. dcta,
IO (1964)376-377
CHIMICA
375
ACTA
A simple and inexpensive method fur the concentration of protein solutions by means of ultrafiltration* We have tested a simple method for the concentration avoids (I) transfer of the material to be concentrated,
of protein solutions which loss of proteins on the
(2)
membrane of an ultr~~filtration apparatus after complete passage of the liquid due to inaccurate timing, (3) loss of proteins caused by denaturation through drying (in evaporation and carbowax methods), and (4) the passage of smal1 molecules into the protein solution from externa1 hypertonic solutions (carbowax method) which may disturb c~~somatagra~hy and/or fingel-printing if enzymatic degradation studies of the protein have to be done. d
a
b
aa
c
e
c
a
glass
plate
b
flier
paper
c
microscoDe
e
dialysis
slide bag
The method requires two glass plates and weights of approximately IO-IS kg. The protein solution to be concentrated is put into a dialysis bag (Visking type, measuring B/32 in diameter was used). This is done in such a way that no spare room is left in the bag. Each end has two sturdy knots. The dialysis bag is then placed on a sheet of filter paper, which in its turn lies on one of the glass plates, Next to the dialysis hag, 4 microscope slides (0.8 mm, thick) are placed, z on each side. Another sheet of filter paper covers the bag and slides. The second glass plate goes on top of it and the weights are placed on top (Fig. I). A significant loss of fluid from the bag will be observed by 4 h. The rate of ultra~ltr~tion is maintained by keeping the bag tense. This is easily accomplished by twisting the bag, beginning at one end. The twisting wiil squeeze the remaining Auid into a single section. A knot is tied in the twisted, empty portion and the now tense bag is placed under pressure again. The volume can easily be reduced to one-tenth of the original in a4 h. The method is preferably performed in a cold room or refrigerator. Dialysis, if needed, can be done before or after concentration of the protein solution. One or more bags can be “squeezed” simultaneously. In the last case the rate of concentration is the same for each of them and is theoretically independent of their relative volumes. * The wurk was supported HE-04706 and WB 04038.
in part by grants from the United States Public Health Service;
CEB?l. Chitn. Acra, 10 (rc364)37j-376
376
SHORT
COMMUNICATIONS
The above mentioned microscope slides are to prevent complete loss of liquid from the dialysis bag. To serve a specific demand, even thinner or also thicker layers may be used. Quantitation of the rate of concentration can be estimated (a) roughly, by measuring and comparing the length of the dialysis bag before and after ultrafiltration (each time in the state of minimum bag volume), (b) more accurately, by weighing the bag before and after concentration (mind wet weight of the bag at the start) or (c) by comparing the O.D.‘s before and at the end of the procedure. Central Laboratory of the Netherlands Red Cross Blood Transfusion Service,
F. PEETOOM PARK
S. GERALD
Amsterdam
(The Netherlands), and Clinical Genetics Laboratory, Children’s Hospital Medical Center, Boston 15, Mass. (U.S.A). Received
April zrth,
1964 Clin.Chinz.
An improved method for calcium determination
Acta, IO (1964)375-376
in serum
The determination of serum calcium according to the FERRO AND HAM method’ using chloranilic acid, is very convenient; however it requires rather large volumes of serum. The method was subsequently modified by the same authors2 by increasing the concentration of chloranilic acid and using microcuvettes for spectrophotometric readings, thus allowing determinations with 0.5 ml of serum. An increase in sensitivity was obtained also by the addition of a 1% FeCl, solution freshly prepared each times. The observation that a sharp increase in optical density of chloranilic acid solutions is obtained at pH 1.5-1.6 (Fig. I), made it possible to increase about fourfold the sensitivity of the method (Fig. z), and thus to operate with 0.5 ml of serum without the need of special apparatuses and using only stable reagents. Reagents (I)
Chloranilic acid solution: I g of chloranilic acid (BDH) is dissolved in 80 ml of water by addition of 7 ml of I N NaOH; the pH is adjusted in a range between 3.5 and 7 by further addition of NaOH or chloranilic acid. The volume is then taken to IOO ml. After 24 h in refrigerator at 4”, the solution is filtered through Whatman No. I filter paper and stored in a brown bottle. Eventual further precipitates
are discarded
by filtration
before use.
(2) Isopropyl alcohol-water 50 : 50 (v/ v). (3) 5% solution of tetrasodium salt of ethylendiaminetetraacetic
acid (BDH).
(4) 0.1 M glycine buffer at PH 1.5. (5) Standard solution of Ca++: 0.2497 g of CaCO, are dissolved in IO ml of I N HCl and taken to 1000 ml with distilled water. A concentration of IO mg Ca++ per IOO ml is thus obtained. Clin. Chim. dcta,
IO (1964)376-377