Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.     

Molecular properties of solubilized CCK receptor from guinea-pig pancreas

In order to characterize the CCK receptor in guinea-pig pancreas, iodinated CCK-39 was bound to pancreatic membranes and the reversible complex was solubilized using various non-denaturing detergents. In term of recovery of ligand stabilized receptors, the relative potencies were Zwittergent 3-14 greater than CHAPS = CHAPSO greater than digitonin greater than MEGA 10 greater than octyl beta-D-glucopyranoside. The stability of receptor complexes was increased by glycerol. Chromatographic analysis revealed that digitonin was the most efficient detergent for disaggregation of CCK receptor complex since it yielded a 76 kDa component in addition to the large components obtained after solubilization with CHAPS and Zwittergent. Furthermore, CCK receptors were covalently labelled using dissuccinimidyl suberate or UV irradiation of labelled membranes by photoactivable radioiodinated CCK-39 and subsequently solubilized by CHAPS + SDS or by SDS alone. A predominant molecule was characterized by chromatography (76 kDa) and SDS-PAGE (89 kDa). In addition to this component, other components having molecular masses of 130-150 kDa, 57 kDa and 40 kDa were detected by SDS-PAGE. They correspond to minor bands. These bands, except the 40 kDa band, were protected from covalent labelling by the presence of CCK-39 (10(-6) M) during initial incubation. Reduction under beta-mercaptoethanol mainly resulted in the decrease of high molecular weight aggregates (Mr greater than 200 kDa). We concluded that for a given detergent a specific molecular weight pattern of solubilized CCK receptor complex is achieved. The minimal component had a molecular mass of 71-84 kDa according to the method of biochemical analysis used.     

Comparisons of plasma membrane polypeptides from soybean and alfalfa

Polypeptides were solubilized with sodium dodecyl sulfate from plasma membrane vesicles of eight varieties of soybean roots [Glycine max (L.) Merr.] and of cultured alfalfa cells (Medicago sativa L.). The solubilized polypeptides were analysed by 2D-polyacrylamide gel electrophoresis. Apparent isoelectric point and MW values were obtained for 80 soybean plasma membrane polypeptides and 44 alfalfa plasma membrane polypeptides. From these data composite distribution patterns were constructed, which are representative of the soybean or alfalfa 2D-gels, respectively. The results showed that the general polypeptide staining patterns were similar for all the soybean varieties, but some minor differences were evident. The alfalfa electrophoretograms differed markedly from the soybean electrophoretograms in specific details, though some general pattern similarities were noted. The data are discussed in terms of a physiological role for the integral plasma membrane polypeptides and in terms of the potential for distinguishing among soybean varieties and between species at the plasma membrane polypeptide level.     

Photosynthate partitioning in split-root citrus seedlings with mycorrhizal and nonmycorrhizal root systems

The plasma membrane ATP-phosphohydrolase (ATPase) from red beet (Beta vulgaris L.) storage tissue was solubilized with the zwitterionic detergent Zwittergent 3-14 from a plasma membrane-enriched fraction which was extracted with the anionic detergent, sodium deoxycholate. For both the extraction of extraneous proteins by deoxycholate and the solubilization of active plasma membrane ATPase by Zwittergent 3-14, the optimal concentration of detergent was 0.1% (weight per volume) with a detergent to protein ratio of 1.0 (milligram per milligram). The properties of the solubilized ATPase were found to be similar to the membrane-bound enzyme with respect to pH optimum, substrate specificity, inhibitor sensitivity, and kinetics of K⁺ stimulation. The solubilized ATPase preparation formed a rapidly turning over phosphoenzyme, the breakdown velocity of which was increased in the presence of 50 millimolar KCl. Solubilization with 0.1% Zwittergent 3-14 following extraction with 0.1% deoxycholate resulted in an increase in both ATPase activity and steady state phosphoenzyme level; however, a direct correspondence between the increase in ATPase activity and phosphorylation level did not exist. It is proposed that this discrepancy may be the result of a detergent-mediated modification of kinetic rate constants in the mechanism of the enzyme.     

Deuterated detergents for structural and functional studies of membrane proteins: Properties,chemical synthesis and applications

Abstract

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.     

Electrophoretic characterization of a detergent-treated plasma membrane fraction from corn roots

Experiments were conducted to determine conditions essential for electrophoretic characterization of a detergent-extracted plasma membrane fraction from corn (Zea mays L.) roots. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) initially gave poor resolution of polypeptides in the plasma membrane fraction and, upon detergent treatment for purification of the proton-pumping adenosine triphosphatase (ATPase), showed no enrichment for a 100 kilodalton catalytic subunit characteristic of the ATPase. In contrast to SDS-PAGE, phenol urea acetic acid (PAU)-PAGE clearly resolved two polypeptides in the 100 kilodalton region that were enriched during detergent treatment and indicated at least one polypeptide forms a phosphorylated intermediate characteristic of the ATPase. Problems with SDS-PAGE were found to be caused, in part, by a combination of endogenous proteases and heat-induced aggregation of high molecular weight proteins. The usually standard procedure of boiling the sample prior to SDS-PAGE caused the aggregation of the 100 kilodalton polypeptides. By controlling for proteases using chymostatin and/or phenylmethane sulfonyl floride, and not boiling the sample prior to electrophoresis, two polypeptides were clearly resolved by SDS-PAGE in the 100 kilodalton region of Triton X-114-extracted membranes from corn, oat, barley, and tomato.     

The solubilization of carotenogenic enzymes of Phycomyces blakesleeanus

The effect of nine ionic and nine non-ionic detergents, over a 0.3–3.0% (w/v) concentration range, on the activity of the enzymes which convert [2-¹⁴C]mevalonic acid into phytoene (7,8,11,12,7′,8′,11′,12′-ψ,ψ-carotene) and β-carotene (β,β-carotene) has been investigated with cell extracts of the C115 carS42 mad-107(?) (β-carotene-accumulating) strain of Phycomyces blakesleeanus. The enzymes catalyzing the conversion of mevalonic acid into phytoene in the C115 and the C5 carB10(?) (phytoene-accumulating) strains of Phycomyces could be released from membranes with high molarity Tris-HCl buffer, but the other carotenogenic enzymes required solubilization with detergents. Enzymic activity was retained with only two ionic detergents (Zwittergents 3–8 and 3–10), whilst Tweens 40 and 60 were the least inhibitory of the non-ionic surfactants. Both Tween 60 and Zwittergent 3–08 solubilized almost 50% of the enzymic activities for the conversion of phytoene to β-carotene, but the former preparation was significantly more stable on storage at ?70°C.     

Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots

The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two-dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements.     

Identification of Tetrahymena Ciliary Surface Proteins Labeled with Sulfosuccinimidyl 6-(Biotinamido) Hexanoate and Concanavalin A and Fractionated with Triton X-114

ABSTRACT. Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from >350 kDa to <20 kDa, were resolved. The major surface 50–60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg²⁺-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.     

Announcement

Recently we described a saturable, high-affinity binding site for vesicular stomatitis virus (VSV) on the surface of Vero cells that appears to mediate viral infectivity. To isolate this binding site, we have extracted Vero cells with the detergent, octyl-β-d-glucopyranoside. The dialyzed detergent extract specifically inhibits the saturable, high-affinity binding of ³⁵S-methionine-labeled VSV to Vero cells. The inhibitory activity is resistant to protease, neuraminidase and heating to 100°C. It is soluble in chloroform-methanol and inactivated by phospholipase C, suggesting that it is a phospholipid. Of various puriifed lipids tested, only phosphatidylserine was capable of totally inhibiting the high-affinity binding of VSV. The half-maximal inhibitory concentration for phosphatidylserine was 1 μM. Phosphatidylserine also inhibited VSV plaque formation by 80%–90%; Herpes simplex virus plaque formation was unaffected. Centrifugation and electron microscopy studies have shown that phosphatidylserine-containing liposomes bind to VSV. The finding that phosphatidylserine directly binds to VSV and inhibits VSV attachment and infectivity suggests that plasma membrane phosphatidylserine could function as a binding site or portion of a binding site for VSV.     

Activation of porcine pancreatic phospholipase A2 by the presence of negative charges at the lipid-water interface

The effect of surface charge on the porcine pancreatic phospholipase A2 catalyzed hydrolysis of organized substrates was examined through initial rate enzyme kinetic measurements. Two long-chain phospholipid substrates, phosphatidylglycerol (PG) and phosphatidylcholine (PC), were solubilized in seven detergents differing in polar head-group charge. The neutral or zwitterionic detergents selected were Triton X-100, Zwittergent 314, lauryl maltoside, hexadecylphosphocholine (C16PN), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The negatively and positively charged detergents used were cholate and CTAB, respectively. In general, the negatively charged phospholipid PG was hydrolyzed much more rapidly than the neutral (zwitterionic) phospholipid PC. The rate of hydrolysis of PG was rapid when solubilized in all the neutral detergents and in cholate but was essentially zero in the positively charged CTAB. Conversely, hydrolysis of PC was negligible when solubilized in neutral detergents, except C16PN, and was maximal in the negatively charged detergent, cholate. The rate of hydrolysis of PC solubilized in a neutral detergent became significant only when a negative surface charge was introduced by addition of SDS. Taken together, these kinetic measurements indicate that the surface charge on the lipid aggregates is an important factor in the rate of hydrolysis of phospholipid substrates and the highest activity is observed when the net surface charge is negative. Fluorescence and electron spin resonance (ESR) spectroscopic data provide additional support for this conclusion. The fluorescence emission spectrum of the single tryptophan of phospholipase A2 is a sensitive monitor of interfacial complex formation and shows that interaction of the protein with detergent micelles is strongly dependent on the presence of a negatively charged amphiphile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitellogenin Receptors From Lobster Oocyte Membrane: Solubilization and Characterization by a Solid Phase Binding Assay

Summary

A solid phase binding assay was developed to study the vitellogenin binding sites from solubilized Homarus americanus oocyte membrane. Different detergents (SDS, CHAPS, DOC) were tested and DOC (sodium deoxycholate) was found to be the most effective agent. The solid phase binding assay involves an adsorption of solubilized membranes in wells of microtitration plates. Enzyme labelling of the ligand was realized by coupling glutaraldehyde treated peroxidase with purified vitellin. Scatchard analysis after competition experiments in different conditions (time and temperature) revealed an apparent equilibrium dissociation constant (Kd) close to 70 nM, reached after one hour incubation at 37°C. Binding activity of oocyte membranes is maximal at the beginning of vitellogenesis and decreases in older oocytes.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Solubilization of yeast plasma membranes and mitochondria by different types of non-denaturing detergents

A comparative study of the solubilization of yeast plasma membranes and mitochondria by different types of non-denaturing detergents has been performed. Zwittergent-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate) at low concentrations (3-4 mM) produced maximum solubilization of both membranes. However, this detergent may inactivate enzymes at high concentrations. Taurodeoxycholate (in the presence of salt) and Triton X-100 were also effective in mitochondria but not in the plasma membranes. Octylglucoside only solubilized these membranes at very high concentrations (20 mM). CHAPS (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) only achieved partial solubilization even at high concentrations. Our results suggest that Zwittergent-14 at low concentrations is one of the most powerful detergents for the general solubilization of native membrane proteins.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

NADH-ascorbate free radical and -ferricyanide reductase activities represent different levels of plasma membrane electron transport

Plasma membranes isolated from rat liver by two-phase partition exhibited dehydrogenase activities for ascorbate free radical (AFR) and ferricyanide reduction in a ratio of specific activities of 1 : 40. NADH-AFR reductase could not be solubilized by detergents from plasma membrane fractions. NADH-AFR reductase was inhibited in both clathrin-depleted membrane and membranes incubated with anti-clathrin antiserum. This activity was reconstituted in plasma membranes in proportion to the amount of clathrin-enriched supernatant added. NADH ferricyanide reductase was unaffected by both clathrin-depletion and antibody incubation and was fully solubilized by detergents. Also, wheat germ agglutinin only inhibited NADH-AFR reductase. The findings suggest that NADH-AFR reductase and NADH-ferricyanide reductase activities of plasma membrane represent different levels of the electron transport chain. The inability of the NADH-AFR reductase to survive detergent solubilization might indicate the involvement of more than one protein in the electron transport from NADH to the AFR but not to ferricyanide.     

Interaction of molybdenum with human erythrocyte membrane proteins

This study describes the interaction of molybdenum with blood components. Molybdenum-99 was added to blood, and after four washings, 3% of the total radioactivity was found in red cells. More specifically, the radioactivity was determined to be associated with the cell membrane. Molybdenum-99 in the +VI form did not interact with the human erythrocyte membrane; however, Mo(V) forms did interact. Of five different compounds, the highes uptake was observed with a brown Mo(V)-ascorbate complex generated from Mo(VI) and ascorbic acid in the molar ratio 1∶20. A membrane suspension of Mo-ascorbate-treated human erythrocytes was prepared and the solubilized proteins were separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). Molybdenum-99 binding to spectrin was demonstrated, as well as some minor interactions with membrane hemoglobin and bands 6 and 8.     

Quantitation of human immunoglobulin G and albumin in electroimmunodiffusion gels containing ionic and nonionic detergents

Quantitation of human immunoglobulin G (IgG) and albumin by agarose electroimmunodiffusion is influenced by the incorporation of ionic and nonionic detergents in the gel. The highest concentrations of each detergent at which human IgG and albumin determinations could be performed without perturbing the quantitations were 4% Triton X-100, 4% Tween 80, 1% NP-40, 0.5% sodium deoxycholate (SDOC), 0.5% Zwittergent, and 0.1% sodium dodecyl sulfate (SDS), and mixtures of Triton X-100, SDOC, and SDS. These detergent combinations all resulted in greater perturbations of albumin quantitation than of IgG. Immunoprecipitation of human IgG was quantitated in the absence and presence of Triton X-100, Zwittergent, and SDS. SDS was shown to cause nonspecific precipitation, whereas below 1% Triton X-100 or 0.5% Zwittergent no effects upon the immunoprecipitations were observed.