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A new method for the concentration of micro protein samples
ANALYTICAL
BIOCHEMISTRY
A New
62, 601-603
Method
(1974)
for the Concentration Protein Samples
of Micro
In many cases before a protein sample can be subjected to analysis by gel filtration, electrophoresis, or immunoelectrophoresis, concentration may be necessary. Some of the commonly used techniques for sample concentration are adsorption on columns, ultrafiltration through cellophane membranes, evaporation of water, absorption by hydrophilic gels, precipitation by salts or organic solvents, and rechromatography. Except for evaporation, all of the other procedures are practical only when the sample size exceeds a few milliliters. Although there is no lower limit on the sample size when evaporation is employed, the resulting increase in salt concentration of the final product is a problem. In this communication we describe a simple technique that is capable of concentrating, severalfold, samples of proteins 25-250 ,pl in volume. The technique is based on the same principle as ultrafiltration through cellophane membranes. METHOD
About 10 g of Aquacide no. 2 (a sodium salt of carboxymethyl cellulose obtained from Calbiochem, Los Angeles, California) were poured evenly on a glass plate 10 X 20 cm. A cellophane sheet, 20 X 30 cm was dipped briefly in distilled water and excess water shaken off. The wet cellophane sheet was layered on the top of the Aquacide bed. The four edges of the cellophane sheet were wrapped around the glass plate and the plate was left to dry for 1 hr at room temperature. As the cellophane sheet dries, it shrinks slightly leaving the aquacide bed firmly sandwiched between the glass plate and the cellophane sheet. At this stage the plate is ready for use. To concentrate a small protein sample, the sample was pipetted as a drop on the cellophane surface. Water and electrolytes are absorbed through the cellophane by the aqua&de. Macromolecules cannot penetrate the cellophane and their concentration in the sample will increase with time. RESULTS
To test the rate of concentration of microsamples by this method, a sample of normal rabbit serum was diluted tenfold with 0.1 M saline Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
601 Inc. reserved.
602
SHORT
0
COMMUNICATIONS
10
20
30 Time
FIG.
1.
FLate of concentration
LO
50
min
of diluted rabbit serum by the described method.
solution. Five aliquots 200 ~1 each of the diluted rabbit serum were pipetted in the form of separate drops on the surface of the aquacide plate. At 10, 20, 30, 40, and 50 min intervals 20 ~1 aliquots were taken from the samples so that each drop was sampled once. The protein content of each 20 ~1 aliquot was determined by the method of Lowry et al. (1) and the results expressed as mg of protein/ml. Figure 1 illustrates the rate of concentration of the diluted rabbit serum over a period of 50 min. It is clear from the figure that the protein sample was concentrated eightfold in about 45 min. The protein recovery after concentration was also tested and was found to vary with sample size and degree of concentration. When a 200 ~1 sample was concentrated fivefold the recovery was about SO%, however when such a sample was concentrated eightfold the recovery dropped to one-half. To test if the process of concentrating serum proteins by this method would adversely affect some of the protein components, a sample of normal human serum was diluted with 0.1 M saline to one-tenth its concentration and then concentrated tenfold by the described method. An aliquot of the concentrated material was analysed by immunoelectrophoresis (2) and the pattern obtained was compared with that of the same serum before it was diluted and concentrated. The immunoelectrophoretic patterns of both sampleswere identical. DISCUSSION
The rate of concentration of any sample by this technique is determined by the area of contact between the sample and the cellophane. If the sample is applied as a drop on the plate the rate of concentration will be slower than if it is applied as a smear but in the second case the recovery after concentration is very poor. For this reason it is always
SHORT
603
COMMUNICATIONS
advisable to let the sample occupy the smallest possible area even though it takes longer to reach the desired concentration. The number of samples that could be concentrated on the same plate depends on the size of the samples and on the dimensions of the plate. A 20 X 30 cm plate could easily accommodate up to 16 samples in two rows. Like many other concentration procedures it is difficult to know exactly when the required concentration is reached without analysing the product. With some experience it is possible to control the final concentration by controling the time. The concentration procedure could be carried out either at room temperature or in the cold and to protect the sample from dust each sample may be covered by a small cup. Once a plate is prepared it could be used immediately or stored for future use. If a plate is partially used it could be stored and the remaining unused part used later. This method has been tested and used for the concentration of protein solutions. It will probably be as useful for the concentration of solutions of other macromolecules such as nucleic acids or polysaccharides. REFERENCES 1. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. J. Biol. Chem. 193, 265. 2. GFCABAR, P., AND WILLIAMS, C. A. (1953) Biochim.
L.,
.~ND RANDALL,
Biophys.
Acta
R.
J.
(1951)
10, 193.
Z. L. AWDEH S. ABU-SAMBA Nutrition Research Laboratory School of Medicine American University of Beirut Beirut, Lebanon Received April 18,1.974; accepted
June
13,197&
BIOCHEMISTRY
A New
62, 601-603
Method
(1974)
for the Concentration Protein Samples
of Micro
In many cases before a protein sample can be subjected to analysis by gel filtration, electrophoresis, or immunoelectrophoresis, concentration may be necessary. Some of the commonly used techniques for sample concentration are adsorption on columns, ultrafiltration through cellophane membranes, evaporation of water, absorption by hydrophilic gels, precipitation by salts or organic solvents, and rechromatography. Except for evaporation, all of the other procedures are practical only when the sample size exceeds a few milliliters. Although there is no lower limit on the sample size when evaporation is employed, the resulting increase in salt concentration of the final product is a problem. In this communication we describe a simple technique that is capable of concentrating, severalfold, samples of proteins 25-250 ,pl in volume. The technique is based on the same principle as ultrafiltration through cellophane membranes. METHOD
About 10 g of Aquacide no. 2 (a sodium salt of carboxymethyl cellulose obtained from Calbiochem, Los Angeles, California) were poured evenly on a glass plate 10 X 20 cm. A cellophane sheet, 20 X 30 cm was dipped briefly in distilled water and excess water shaken off. The wet cellophane sheet was layered on the top of the Aquacide bed. The four edges of the cellophane sheet were wrapped around the glass plate and the plate was left to dry for 1 hr at room temperature. As the cellophane sheet dries, it shrinks slightly leaving the aquacide bed firmly sandwiched between the glass plate and the cellophane sheet. At this stage the plate is ready for use. To concentrate a small protein sample, the sample was pipetted as a drop on the cellophane surface. Water and electrolytes are absorbed through the cellophane by the aqua&de. Macromolecules cannot penetrate the cellophane and their concentration in the sample will increase with time. RESULTS
To test the rate of concentration of microsamples by this method, a sample of normal rabbit serum was diluted tenfold with 0.1 M saline Copyright All rights
@ 1974 by Academic Press, of reproduction in any form
601 Inc. reserved.
602
SHORT
0
COMMUNICATIONS
10
20
30 Time
FIG.
1.
FLate of concentration
LO
50
min
of diluted rabbit serum by the described method.
solution. Five aliquots 200 ~1 each of the diluted rabbit serum were pipetted in the form of separate drops on the surface of the aquacide plate. At 10, 20, 30, 40, and 50 min intervals 20 ~1 aliquots were taken from the samples so that each drop was sampled once. The protein content of each 20 ~1 aliquot was determined by the method of Lowry et al. (1) and the results expressed as mg of protein/ml. Figure 1 illustrates the rate of concentration of the diluted rabbit serum over a period of 50 min. It is clear from the figure that the protein sample was concentrated eightfold in about 45 min. The protein recovery after concentration was also tested and was found to vary with sample size and degree of concentration. When a 200 ~1 sample was concentrated fivefold the recovery was about SO%, however when such a sample was concentrated eightfold the recovery dropped to one-half. To test if the process of concentrating serum proteins by this method would adversely affect some of the protein components, a sample of normal human serum was diluted with 0.1 M saline to one-tenth its concentration and then concentrated tenfold by the described method. An aliquot of the concentrated material was analysed by immunoelectrophoresis (2) and the pattern obtained was compared with that of the same serum before it was diluted and concentrated. The immunoelectrophoretic patterns of both sampleswere identical. DISCUSSION
The rate of concentration of any sample by this technique is determined by the area of contact between the sample and the cellophane. If the sample is applied as a drop on the plate the rate of concentration will be slower than if it is applied as a smear but in the second case the recovery after concentration is very poor. For this reason it is always
SHORT
603
COMMUNICATIONS
advisable to let the sample occupy the smallest possible area even though it takes longer to reach the desired concentration. The number of samples that could be concentrated on the same plate depends on the size of the samples and on the dimensions of the plate. A 20 X 30 cm plate could easily accommodate up to 16 samples in two rows. Like many other concentration procedures it is difficult to know exactly when the required concentration is reached without analysing the product. With some experience it is possible to control the final concentration by controling the time. The concentration procedure could be carried out either at room temperature or in the cold and to protect the sample from dust each sample may be covered by a small cup. Once a plate is prepared it could be used immediately or stored for future use. If a plate is partially used it could be stored and the remaining unused part used later. This method has been tested and used for the concentration of protein solutions. It will probably be as useful for the concentration of solutions of other macromolecules such as nucleic acids or polysaccharides. REFERENCES 1. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. J. Biol. Chem. 193, 265. 2. GFCABAR, P., AND WILLIAMS, C. A. (1953) Biochim.
L.,
.~ND RANDALL,
Biophys.
Acta
R.
J.
(1951)
10, 193.
Z. L. AWDEH S. ABU-SAMBA Nutrition Research Laboratory School of Medicine American University of Beirut Beirut, Lebanon Received April 18,1.974; accepted
June
13,197&