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A simple assay for quantification of protein in tissue sections,cell cultures, and cell homogenates, and of protein immobilizedon solid surfacesAngela Dieckmann-Schuppert1, Hans-Joachim Schnittler2

1 Zentrum für Hygiene und Medizinische Mikrobiologie, Arbeitsgruppe Parasitologie, Philipps-Universität Marburg,Robert-Koch-Strasse 17, D-35037 Marburg, Germany2 Anatomisches Institut der Julius-Maximilians Universität Würzburg, Koellikerstrasse 6, D-97070 Würzburg, Germany

&misc:Received: 9 August 1996 / Accepted: 31 October 1996

&p.1:Abstract. The determination of total protein is often akey step for the quantitative analysis of various parame-ters in tissue and general biochemical research. The clas-sical protocols are restricted to a few compatible buffers,and protocols for the determination of protein in solu-tions containing protein agglomerates or of protein im-mobilized on solid surfaces are not available. In suchcases, quantification may be complicated. Here, we de-scribe a simple sensitive method for protein quantifica-tion circumventing all these restrictions. Proteins in so-lution or suspension in any buffer are spotted onto cellu-lose acetate, dried, and stained with Amido Black. Afterwashing off the excess dye, bound Amido Black is solu-bilized in an acidic solution and determined photometri-cally. Tissue slices (fixed or native), adherent cell cul-tures, or Western blots can also be stained and their pro-tein content determined irrespective of the supportingmaterial. A micro-version of the protocol for proteins insolution allows large numbers of samples to be evaluatedat a time in microtitration plates and requires only1–2 µl per sample. A linear concentration dependency(r2=0.950–0.999) was obtained for all samples in allcases investigated. The method presented here permitsthe exact determination of soluble protein in a large vari-ety of buffers, of insoluble or immobilized protein pres-ent on a wide variety of supports, and even of wholecells or tissue slices.

&kwd:Key words: Protein determination – Quantification intissue slices and cell cultures – Detergents – High saltconcentrations – Solid surfaces – Western blot

Introduction

The quantification of a specific biochemical signal, suchas an antibody reaction, the activity of an enzyme, or theincorporation of radioactivity is a central task in anatom-ical, cell biological, biochemical, and pathogenetic re-search. Although highly sensitive indicator methods de-tecting extremely low levels of the respective signals, forexample, enhanced chemiluminescence, or the use of ra-dioactive substances, are available, their specific quanti-fication in tissue sections, adherent or suspended cellcultures, cells grown on biomaterials, tissue homoge-nates, or on Western blots is often limited, because of aninsufficient determinability of total sample protein. Amethod allowing the precise determination of protein notonly in solution, but also in these special cases, and re-quiring as small a sample as possible would therefore beof great interest.

The classical, widely used protocols for protein deter-mination, such as the Lowry (Lowry et al. 1951) orBiuret (Weichselbaum 1946) methods and all their modi-fications, are applicable only to solutions. Moreover,these methods suffer from the limitation that they are notcompatible with certain buffers, high salt concentrations,or the presence of detergents. In order to avoid these dif-ficulties, a precipitation step is often included, whichmay however not always be quantitative and reproduc-ible, especially in dilute solutions. The classical methodsface even more restrictions when the protein to be deter-mined presents itself as aggregates, whole cells, orbound to solid surfaces, i.e., in the cases mentionedabove. Under these circumstances, protein determinationproves extremely complicated and often the amount ofprotein can only be roughly estimated.

The concept of staining protein spots dotted onto asolid support with Amido Black followed by elution andphotometric quantification of the dye was put forwardover thirty years ago (Heinzel et al. 1965), but has so farnot gained wide propagation. This method has attractedmore interest following the publications of Schaffnerand Weissmann (1973) and Henkel and Bieger (1994),

This work was supported by the Deutsche Forschungsgemein-schaft, SFB 355/B5

Correspondence to:Hans-J. Schnittler (Tel.: +49–931–312706;Fax: +49–931–15988; E-mail: anat020@rzbox.uni-wuerzburg.de)&/fn-block:

Cell Tissue Res (1997) 288:119–126

© Springer-Verlag 1997

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because detergents such as SDS do not interfere with thedetermination. Sportsman and Elder (1984) have sug-gested circumventing the required photometric evalua-tion by the use of a laser gel scanner, but this equipmentmay not be available in every laboratory.

Here, we present a modified improved method ofAmido Black protein determination that exhibits highsensitivity not only in buffers containing detergent andhigh salt, but also with respect to tissue sections, cell cul-tures, cell and tissue homogenates, and protein immobi-lized on solid surfaces. We believe that the protocols pro-vided here will be useful for every laboratory where sen-sitive protein quantification is a part of the daily routinebut still an “art” depending on the respective problem.

Materials and methods

Materials and instruments

Cellulose acetate (CA) sheets measuring 25×160 mm were ob-tained from Schleicher and Schüll (Dassel, Germany). Nitrocellu-lose (NC) was obtained from the same manufacturer and cut intopieces of a corresponding size. Phosphate-buffered saline (PBS)containing 8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na2HPO4, 0.2 g/lKH2PO4 (pH 7.4) was obtained from Seromed (Berlin, Germany).Bovine serum albumin (BSA) was from Sigma (Deisenhofen,Germany) and Amido Black 10B from Merck (Darmstadt, Ger-many). All other chemicals were of the highest purity available.Polystyrene microtitration plates and multiwell cell-culture disheswere from by Becton Dickinson (Heidelberg, Germany). Cryostatsections were cut on a Frigocut 2800 (Reichert-Jung, Nußloch,Germany). Photometric analysis was performed on a Pharmacia Ul-traspec 4 photometer (Pharmacia, Freiburg, Germany), and spectrawere recorded with a Spekol VIS (Zeiss, Jena, Germany) automaticspectrophotometer. A Titertek Multiskan Plus MKII enzyme-linkedimmunosorbent assay (ELISA) reader (Eflab, Helsinki, Finnland)was used for the photometric evaluation of samples in microtitra-tion plates. Image analysis of gels and the determination of tissuesections was performed by using the gel-blotting macro or areacount of NIH-Image version 1.52 on a Macintosh computer. Sus-pended cells were counted and analyzed in a CASY-1 cell counterand analysis system (Schaerfe System, Reutlingen, Germany).

Tissue treatment and sections

Livers from rats were removed after decapitation or after perfu-sion fixation with 4% formaldehyde in PBS under deep pentobar-bital anesthesia via the left heart ventricle as described in detailelsewhere (Asan 1993). They were dissected into small pieces andfrozen at –20°C. Cryostat sections (5µm in thickness and approx-imately 3×4 mm) were cut, mounted on polyornithine-coated cov-erslips, and dried at 60°C for 1 h.

Cells

Human umbilical vein endothelial cells (HUVEC) were isolatedand cultured on 6-cm2 glass slides as described elsewhere(Schnittler et al. 1993). For the experiment shown in Fig. 6, thesecells were fixed in 2% formaldehyde in PBS, followed by 3 wash-es in PBS, for 10 min.

Buffers tested

BSA stock solutions and dilutions were prepared in the followingsolutions: bidistilled water, PBS, 4 M urea in PBS, 1.5 M KI in

PBS, and sample buffer for polyacrylamide gel electrophoresiscontaining 6% (w/v) SDS, 9% (v/v) glycerol, 10 mM dithiothre-itol, 0.187 M TRIS-HCl at pH 6.9, supplemented with 0.02%(w/v) bromophenol blue, 1% Triton X-100, or 1% NP-40 whereindicated. All samples in sample buffer were heated to 95°C for5 min and stored at –20°C until used.

Dissolution protocol for protein quantification

Macro-method. &p.2:Fields of 1 cm width were marked on CA or NCsheets with a pencil. The sheets were then mounted horizontally ina holder between magnets. Defined volumes of protein samples(10 µl) were applied onto each field. Blanks containing only therespective buffer served as the zero control. The sheets were driedfor 15 min at room temperature (RT) and subsequently immersedin the staining solution Istain [0.5% (w/v) Amido Black 10B, 45%(v/v) each of methanol and water, and 10% glacial acetic acid] for10 min. Staining was followed by three washes in solution Iwash(47.5% each of methanol and water, and 5% glacial acetic acid)for 5 min each. The sheets were then dried again (5 min at RT),the individual samples cut apart, and placed into separate testtubes (2 ml Eppendorf cups). Dissolution of the CA pieces wasthen performed in 1 ml dissolution solution Idiss (80% formic acid,and 10% each of glacial acetic acid and trichloroacetic acid) byincubation at 50°C for 15 min or 30 min at RT under shaking. Theresulting blue solutions were read photometrically at 620 nm or672 nm, respectively, against the corresponding reagent blank. Allanalyses were performed in triplicate or quadruplicate, and linearregression lines were calculated.

Micro-method. &p.2:CA discs of 5.5 mm diameter were placed into mi-crotitration plates (96 well), and 1 or 2µl standard or sample pro-tein were spotted onto the CA and further processed as describedfor the macro-method. Dissolution solution (100 or 200µl) wasused to dissolve the CA paper after the staining and destainingprocedure. The microtitration plates were covered doubly withParafilm to avoid evaporation of the solution. The samples wereshaken for 30 min at room temperature, and the absorbance wasmeasured at 620 nm.

Elution protocol for protein quantification

The method was performed according to Henkel and Bieger(1994) as follows: staining for 3 min in solution IIstain [0.1% Ami-do Black 10B (w/v), 45% (v/v) each of methanol and water, 10%glacial acetic acid], 2 washes for 3 min in water, then 2 washes for3 min in solution IIwash (90% methanol, 2% acetic acid, 8% wa-ter), and again for 5 min in water. Elution of the dye from thedried sheets was accomplished by 1 ml eluent solution IIelut (50%ethanol, 50% 50 mM NaOH/0.1 mM EDTA) per sample undershaking for 30 min. Where indicated, a combination of the disso-lution and elution protocols was performed.

Electrophoresis and blotting

SDS-polyacrylamide slab gel electrophoresis, electroblotting ontonitrocellulose, and fixation-staining of the slab gels with Coomas-sie blue were performed according to standard procedures as de-scribed elsewhere (Schnittler et al. 1990).

Results and discussion

Staining of proteins by Amido Black, spectra,and standards

The adsorption of Amido Black to any protein is stoichio-metrically dependent on the number of free amino groups

present in, and accessible on the given protein. This al-lows the determination of protein in the presence of chao-tropic salts and/or detergents as well as in any physicalstatus of the protein (i.e., dissolved, suspended, in mem-branes of membrane bound proteins, or in tissue slices).

To investigate the pH-dependence of the AmidoBlack absorption, spectra were run in the acidic dissolu-tion solution (Idiss) and compared with spectra recordedin the basic elution solution (IIelut), each against the cor-responding solutions alone. The concentrations of Ami-do Black tested was between 3,25µg/ml and 15µg/ml.An isosbestic point is present at 620 nm (Fig. 1), render-ing absorbance values recorded at this wavelength di-rectly and quantitatively comparable, and excluding anypH-effect. Maximal absorbance in the acidic solutionIdiss is, however, found at 672 nm, exceeding the valuefound at 620 nm by 27%. Nevertheless, all spectrophoto-metric recordings documented here were taken at620 nm for the sake of comparability. It should beemphasized, however, that reading at 672 nm will fur-ther increase the sensitivity of determinations by about25%.

BSA has been shown to be a suitable representativeprotein for Amido Black staining (Heinzel et al. 1965;Henkel and Bieger 1994). Proteins with extreme isoelec-tric points will stain better or worse, corresponding tothe number of free amino groups present. If such excep-tional proteins are to be determined by the Amido Blackmethod, then that particular protein or at least a similarone should be used for the generation of a standardcurve. This consideration is, however, independent of theother advantages of the Amido Black method.

Quantification of protein, protein-aggregates,and suspended cells in various buffers

Aliquots of samples containing protein, protein-aggre-gates (e.g., homogenized tissue), or whole suspended

cells can be spotted onto CA, air dried, stained and, de-stained by using the CA-dissolution protocol as de-scribed above. In our first series of experiments, we in-vestigated the influence of various buffers containinglarge amounts of salts and/or detergents on the sensitivi-ty of the protein determination.

Influence of buffer composition on the sensitivity of theCA-dissolution protocol. &p.1:Standard dilution series rang-ing from 1.25–40µg/10 µl (i.e. 0.1–4.0 mg/ml) wereprepared for all eight buffers listed in the Methods sec-tion. All the resulting regression lines exhibited correla-tion coefficients (r2) of 0.990 or above and showed simi-lar slopes ranging from 2.0 to 2.8 [10×OD×ml/mg] (Ta-ble 1). The sensitivity of the method therefore does notvary greatly with the chemical composition of the re-spective buffer, rendering the method suitable for an ex-tremely wide range of applications. The slight variationin the response factors may be a result of the differentdegree of protein unfolding in the respective buffers,caused by the different denaturing power of these buf-fers. The excellent performance of the CA-dissolutionprotocol in all sample buffers is probably attributable tothe removal of most, if not all, buffer components bywashing with solution Iwash, which contains water andmethanol in equal parts but is still not acidic enough toremove the Amido Black from the protein (pH Iwash=2.5;but Idiss<0).

Determination of protein in solutions containing protein-aggregates, cell fragments, whole cells, or small piecesof tissue fragments. &p.1:Cell homogenates, suspended wholecells, or small tissue fragments can be dropped directlyonto CA and their protein content exactly determined bythe CA/dissolution protocol as described for soluble pro-teins above. A typical result is shown in Fig. 2, wheresuspended hybridoma cells at a very low density wereused. This method is therefore applicable for the deter-mination of protein in suspended cells and for the reli-

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Fig. 1. Spectra of Amido Black 10B (12µg/ml) in the acidic dis-solution solution Idiss (dotted line) and in the alkaline elution solu-tion IIelut (continuous line). Note the isosbestic point at 620 nmand the absorbance maximum of the acidic sample at 672 nm&/fig.c:

Table 1. Slopes of the regression lines obtained for BSA(1.25–4 mg/ml) in various buffers as obtained by the CA-dissolu-tion method, and corresponding regression correlations. Note thatthe slopes increase with increasing denaturing power of the re-spective buffer (r2, square of correlation coefficient from linear re-gression analysis)&/tbl.c:&tbl.b:

Buffer Slope r2

Distilled water 2.03 0.997PBS 2.18 0.996Sample buffer 2.37 0.996

+ 1% Triton X-100Sample buffer 2.47 0.996

+ 1% Triton X-100 + 1%NP-40

Sample buffer 2.48 0.996+ bromophenol blue

Sample buffer 2.49 0.9944 M urea 2.76 0.9901.5 M KI 2.81 0.980

&/tbl.b:

wavelength [nm]

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able indirect estimation of cell numbers, in cases wherea cell counter is not available. The limiting factor forprotein determination in solutions containing cells, cellaggreggates, or tissue fragments is mechanical adhesiononto CA. Small flat pieces of not more than 2–3 mm2

stick much better than do cubic pieces. Thus, tissueshould be homogenized as much as possible, when pro-tein determination is to be performed in solutions inwhich tissue fragments are suspended.

A micro-version of the CA-dissolution protocol applica-ble to microtitration plates. &p.1:The CA-dissolution protocolcan be performed on mini-discs of CA measuring5.5 mm in diameter and placed into individual wells of96-well flat-bottom microtitration plates. In this case,the sample volume should be reduced to 1–2µl and thatof the dissolution solution to 100–200µl (Fig. 3). Thesamples are applied to and dried on the CA discs directlyin the micro-wells. Staining, destaining, and dissolutionsolutions can be dispensed and aspirated by means ofstandard ELISA-processing instruments. Corresponding-ly, spectrophotometric evaluation of the microtitrationplates can be achieved with an automatic ELISA reader.The polystyrene plates are not affected by the stronglyacidic solution Idiss, even if they are exposed over severaldays.

Performance of the Amido Black method on CA com-pared with NC. &p.1:The use of NC allows the determinationof solubilized protein by Amido Black binding in thepresence of SDS with an elution protocol (Henkel andBieger 1994). We have compared the performance of themethod described here with the elution protocol by Hen-kel and Bieger (1994).

Dilution series of BSA in SDS sample buffer contain-ing bromophenol-blue were prepared as above in qua-druplicate and subjected in parallel to the dissolutionand to the elution protocol. Whereas NC, as already not-

ed by others (Henkel and Bieger 1994), softened andbecame difficult to handle following the elution protocol(the critical step being the wash in solution IIwash), theCA paper was easy to handle throughout all proceduresof the dissolution protocol.

When NC was used instead of CA in the dissolutionprotocol, background staining was much higher than onCA. At 620 nm, background staining ranged from 0.35to 0.40 on NC, but was only 0.020 to 0.025 on CA. Sub-traction of the high background level rendered the deter-mination on NC less sensitive than that on CA. More-over, NC did not dissolve during the dissolution step(Idiss), but the dye itself came off completely after exces-sive shaking. Comparing the regression lines shown inFig. 4, it is evident that the dissolution protocol appliedto CA has the highest sensitivity and exhibits excellentcorrelation (r2 = 0.999). This is of great importancewhen small amounts of protein (in the range of 0.5 up to50 µg total protein) are to be determined.

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Fig. 2. Application of the Amido Black method to the determina-tion of protein in cultured suspended hybridoma cells (660cells/µl). An excellent correlation is obtained between the numberof suspended hybridoma cells and the absorbance measured afterdrying of the cell suspension onto CA filters&/fig.c:

Fig. 3a, b.Application of the CA-dissolution protocol to microti-tration plates. a Photograph of a plate with triplicate samples tak-en after treatment with solution Idiss. The absolute amounts of pro-tein correspond to the relative amounts indicated in b. b Linear re-gression line obtained from the experiment in a using 2-µl sam-ples of BSA (0.125–4 mg/ml) in electrophoresis sample buffer in-cluding bromophenolblue. The CA pieces were dissolved with200 µl solution Idiss. Note that the path length in microtitrationplate photometry is different from that in standard cuvettes (1 cm)and that therefore the absorbance is also different from thoseshown in Fig. 4&/fig.c:

When CA was used instead of NC in the elution pro-tocol, the slope was reduced compared with the CA-dis-solution protocol. When CA was immersed in the elu-tion solution IIelut for Amido Black removal, the individ-ual strips tended to disintegrate rendering the final solu-tion cloudy. This turbidity could be removed by centrifu-gation, but the sensitivity, as expressed by the slope ofthe regression line, was not improved (Fig. 4).

Taken together, these results show that the determina-tion of proteins on NC tends to be less sensitive than onCA. Where the use of NC is unavoidable, however, theelution protocol seems more suitable. We suggest thatthe lower sensitivity of both CA and NC in the elutionmethod compared with the CA-dissolution protocol iscaused by an unspecified loss of protein in the washingsolution IIwash. Additionally, we have observed thatequal amounts of protein dissolved in equal volumes ofbuffer, when applied to both CA and NC, are distributedmore evenly on CA than on the same area of NC. Thus,a local protein overloading of NC and a correspondingloss of protein, especially at higher concentrations,should be expected to occur more easily on NC than onCA. Therefore, CA should be used wherever possible.

If the micro-version of protein determination is to beperformed, the use of NC is not recommended, since NCdissolves neither in elution solution (IIelut) nor in the dis-solution solution (Idiss). NC discs would therefore haveto be individually removed from the microtitration plateprior to photometric evaluation, rendering the wholeprocedure tedious and not applicable for the evaluationof many samples. Moreover, the removal of the NC discwill also cause unequal reduction of fluid sticking to theNC, thereby reducing the accuracy of the test. In con-trast, the micro-variant of the CA-dissolution protocol

could well be automated and thus be reliably performedon many samples at a time. It should be extremely suit-able, for example, in the process of monitoring column-chromatographic eluents, especially when harsh buffersare used. Furthermore, the protocol is not limited regard-ing time and can be interrupted for hours after every stepand continued later.

Quantification of protein presentas protein-aggregates, adherent cells,or tissue sections immobilized on solid surfaces

Using the easy-to-handle Amido Black staining protocolfor the determination of proteins in solution, we testedwhether this protocol is applicable to protein quantifica-tion in cases where proteins, protein aggregates, adher-ent cells, or whole tissue sections are immobilized orgrown on various solid surfaces.

Quantitative evaluation of Western blots. &p.1:Western blotanalysis is not only used for qualitative, but also forquantitative evaluation of specific proteins. One of theproblems associated with quantitative Western blot anal-ysis is the unknown amount of protein present on the ni-trocellulose following the electrotransfer. Here, we showthat protein which has been electroblotted onto NC canbe accurately and directly determined on the NC sheetsby Amido Black staining. Several different concentra-tions of a standard protein and of the samples to be ana-lyzed should be used and blotted in parallel in order tocorrect for the transfer rate of the individual experimentand for the binding rate of the respective protein, whichis known to be affected by its hydrophobicity. Imageanalysis of identical Coomassie-stained gels loaded withthe same amount of sample, either not subjected to elec-troblotting or after electroblotting, indicates a transferrate of approximately 50% for the particular gel shownin Fig. 5a. However, the correlation is unsatisfactory foran exact quantification. Amido Black determination ofthe NC-blotted proteins compared with the originallyloaded amount reveals a comparable transfer rate (58%)but a significantly higher correlation (Fig. 5b), which al-ways exceeded 0.95 for three independent experiments.Both protocols (elution and dissolution) can be used forprotein determination, but, for the sake of more conve-nient handling of the NC, the data shown in Fig. 5b wereobtained by using the NC/dissolution protocol. If, how-ever, the amounts of protein to be determined are verysmall or if single bands are to be quantified, elutionshould instead be preferred for the reasons given before.Thereby, the determination of as little as 0.1µg proteinin a single band has been achieved with only 100µl ofdissolution solution Idissor elution solution IIelut.

Determination of protein in adherent cultured cells. &p.1:An-other problem that can easily be solved by the presentedmethod is the determination of protein in cultured adher-ent cells in cases where other quantifications are to beperformed on the same cells prior to or after this step.As shown in the example in Fig. 6, adherent cells are

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Fig. 4.Linear regression lines obtained for standard dilution series(0, 0.125, 0.25, 0.5, 1.0, 2.0, and 4.0 mg/ml) applied in quadrupli-cate to CA or NC sheets and treated according to the dissolutionand the elution protocol. For the sake of distinctness, the individu-al data points are not displayed. Squares of the regression coeffi-cients are: 0.999 (CA/diss.), 0.993 (CA/el.), 0.998 (NC/el.), and0.997 (NC/diss.). Note that the slopes of the standard lines reflectthe determination sensitivity&/fig.c:

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also suited for Amido Black staining. Endothelial cells(HUVEC) were seeded in triplicate at different densitiesonto polystyrene 24-well culture dishes and fixed by 2%formaldehyde after incubation for 12 h. To obtain the da-ta in Fig. 6, it was necessary, in the case of the two high-est cell densities, to dilute the solution prior to spectro-photometric evaluation. Corresponding results were ob-tained with cells grown on glass coverslips (data notshown). Simultaneously, standard protein was spottedonto polystyrene culture dishes or glass coverslips, re-spectively, dried at 60°C for 1 h, and subsequently pro-cessed for Amido Black staining by the dissolution pro-tocol.

An Amido Black staining protocol has recently beenpublished for quantifying the amount of protein in ad-herent cultured cells (Schulz et al. 1994). In contrast to

the method presented here, an alkaline elution solution isused to remove bound Amido Black for photometricquantification. In our experience, alkaline treatment ofimmobilized protein on solid surfaces causes a substan-tial loss of protein (see also below). Thus, if two inde-pendent parameters are to be determined within the samecells (e.g., incorporation of radioactivity and totalamount of protein), the acidic dissolution protocolshould be preferred to avoid protein loss.

The differences in the amount of protein betweenHUVEC (Fig. 6) and hybridoma cells (Fig. 2) with re-spect to cell number are caused by the different sizesand therefore different amounts of protein in the respec-tive cells. HUVECs have a volume of approximately3000 fl, whereas that of the hybridoma cells is onlyabout 500 fl.

Application to tissue sections. &p.1:Cryostat sections (5µm inthickness) of fixed or native (but not embedded) tissuewere immobilized on polyornithine-coated coverslips,dried for 1 h at 37°C, and subsequently processed ac-cording to the dissolution protocol as described above.As shown in Fig. 7a, an excellent linear correlation wasobtained between the number of sections and the absor-bance measured after dye elution. To evaluate these datafurther, the areas of the tissue sections were measured byimage analysis and correlated with the number of sec-tions. As shown in Fig 7b, a good correlation existed be-tween the number of sections and the tissue-section ar-eas.

We have not determined the time course of stainingand destaining but we assume that it will follow the dif-fusion law and be dependent on convection (shaking).For 5-µm fixed tissue sections, a duration of 10 min foreach step is sufficient. Increased duration of staining ordestaining for up to 2 h does not affect the absorbanceobtained after dissolution in Idiss. We should like to pointout that special care has been taken in these experiments

Fig. 5a,b.Quantitative evaluation of Western blots. a Coomassie-blue-stained polyacrylamide slab gels used for the separation oftotal extracts of cultured endothelial cells dissolved in SDS sam-ple buffer (5, 10, and 20µg total protein) were subjected to imageanalysis without (upper line) and following (lower line) Westernblotting. The comparison of the slopes shows a transfer rate of ap-proximately 54%, but the correlation coefficients r2 are low. b De-termination of protein of the corresponding blot on NC by the dis-solution protocol. Spectrophotometric quantification of the boundAmido Black (lower line) compared with the amount of proteinoriginally applied to the gel (separately spotted onto NC directly,upper line) reveals a similar transfer rate (58%) but exhibits a sig-nificantly improved correlation&/fig.c:

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Fig. 6. Protein determination of adherent endothelial cell culturesof different cell densities in a 24-well polystyrene culture dish bythe dissolution protocol. Spectrophotometric data show a goodcorrelation between the number of cells and the resulting absor-bance&/fig.c:

to obtain slices of the same size and thickness. This can-not be guaranteed when different tissue samples are be-ing compared. Thus, the accuracy of the results will de-crease when the parameters measured can only be relat-ed to the tissue volume estimated from an area count byusing image analysis.

The absorbance can be standardized for all applica-tions of protein determination on solid surfaces by dry-ing known amounts of protein on the appropriate sur-face and subjecting these to the same protocol. An ex-ample in which coverslips were coated with definedamounts of protein, fixed over vaporizing formalde-hyde, and further analyzed by the dissolution protocol isshown in Fig. 7. Fixation over vaporizing formaldehydeis preferred to immersion of the samples in a solution,because protein may dissociate from the surface, evenin the presence of formaldehyde. Unfixed samples ana-lyzed in parallel revealed the same results (data notshown), indicating that protein determination by AmidoBlack is not influenced by crosslinking of the proteins(fixation). A loss of protein from the surfaces when us-ing the Amido Black staining solution does not occur,because the acidic solution precipitates the protein ontothe surface.

Possibility of subsequent double determinations on thesame sample. &p.1:Samples immobilized on glass (e.g., cov-erslips) or plastic (e.g., polystyrene culture dishes) sur-faces, from which bound Amido Black is desorbed bythe use of solution Idiss, can subsequently be subjected toanother cycle of staining and destaining yielding nearlythe same absorbance, thereby suggesting that no proteinhas been lost. Absorbances measured after a secondstaining/destaining cycle (samples of cultured HUVEC)differ by less than 8% from those obtained in the firstcycle. For this application, we recommend crosslinkingthe immobilized proteins by treatment with formalde-hyde. When, however, the alkaline solution Ielut is usedto extract the dye from the sample, a substantial loss ofprotein occurs in the subsequent staining cycle. This lossunder alkaline conditions is probably caused by a muchhigher rate of partial sample disintegration and desorp-tion, together with an eventual partial hydrolysis. There-fore, the use of the acidic solution Idiss for dye removalallows subsequent determinations of other parameters onthe same sample. This holds for a variety of applica-tions. One example is the treatment of cells or tissuewith aggressive buffers to extract small molecules (e.g.,extraction of ATP from cells or tissue in models of hyp-oxia or ischemia of the heart), with the aim of correlat-ing the respective parameter to total sample protein. Fur-thermore, the application of the Amido Black methodshould also allow a reliable determination of antigen-an-tibody reactions (e.g., receptor densities) or of enzymeactivities in tissue sections. Moreover, the determinationof radioactivity will encounter neither color nor chemi-cal quenching after removal of the dye itself and of theotherwise strongly quenching acid solution Idiss. Thus,double determinations of protein content and other cellbiological parameters can be rendered much more accu-rate and reliable.

Range of applications for the determination of proteinon solid surfaces. &p.1:Preliminary results have shown that themethod presented here not only allows the quantificationof protein bound to glass or culture-dish surfaces, but alsothat of protein immobilized on chromatographic matricesor micro-beads used for cell culture, and the determina-

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2

2ab

sorb

ance

(62

0n

m)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.00 1

1

2

2

3

3

4

4

no. of sections

no. of sections

r

r

r

0.971

0.978

0.994

a

b

c

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.00

area

[mm

]

0protein [µg]

10 20 30 40

abso

rban

ce(6

20

nm

)

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Fig. 7a–c.Application of the Amido Black method to whole tis-sue sections. Cryostat sections of formaldehyde-fixed rat liver im-mobilized on glass coverslip were analyzed by the dissolutionprotocol. a Correlation between the number of sections and the re-sulting absorbance per coverslip. b Correlation between the num-ber of sections and their area obtained by image analysis. c Stan-dardization by known amounts of BSA (in PBS) dried onto simi-lar coverslips&/fig.c:

126

tion of protein present on disposable medical latex prod-ucts (e.g., gloves), which have been shown to contributeto the development of allergies (Yunginger et al. 1994). Afurther important application will be the determination ofprotein adsorbed onto biomaterials. Further experimentsin this field are currently being performed.

Conclusions

The data presented here demonstrate that Amido Blackstaining is a valuable method that is simple to perform.It can be applied to a wide variety of solutions and buf-fers, including those containing detergents and/or highconcentrations of salts. Most importantly, protein can bedetermined in tissue sections and cell cultures, and evenwhen protein aggregates, including whole cells or tissuefragments, are present in solution. The determination ofprotein can also be performed when proteins are cross-linked by formaldehyde, irrespective of whether the pro-tein is solubilized, suspended, or immobilized on solidsurfaces.

&p.2:Acknowledgements.We thank Mrs. Martina Koch for skillful tech-nical assistance. Dr. F. Schmitz, Anatomisches Institut Würzburg,kindly provided the hybridoma cells.

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