CS277303B6

CS277303B6 - Method of protein detection by silver detection

Info

Publication number: CS277303B6
Application number: CS886347A
Authority: CS
Inventors: Kamil Ing Csc Marcinka
Original assignee: Virologicky Ustav Sav
Priority date: 1988-09-26
Filing date: 1988-09-26
Publication date: 1993-01-13
Also published as: CS634788A3

Abstract

Platňový gél sa po elektroforéze perle vo fixačnovymývacom roztoku, potom 25 až 50 % metanolom alebo etanolom a vodou, ďalej sa naň pósobí roztokom tiosíranu sodného, po opláknutí vodou následuje pósobenie farbiacim roztokom, tvořeným dusičnanom striebomým. Po krátkom praní vodou následuje pósobenie vývojkou, při ktorej dochádza k vizualizácii bielkovín v géli. Vyvolávací proces sa zastaví zastavovacím kúpeíom a gél sa nakoniec perle vodouAfter electrophoresis, the plate gel is rinsed in a fixative-elution solution, then 25 to 50% methanol or ethanol and water, then treated with a sodium thiosulfate solution, rinsed with water, followed by treatment with a staining solution consisting of silver nitrate. After a short wash with water, treatment with a developer follows, during which the proteins in the gel are visualized. The development process is stopped with a stop bath and the gel is finally rinsed with water.

Description

Spósob stanovenia bielkovín detekčnou technikou striebromMethod for determination of proteins by silver detection technique

Ohlasů technikyTestimonials technique

Vynález sa týká spósobu stanovenia bielkovín detekčnou technikou striebrom. 'The invention relates to a method for the determination of proteins by a silver detection technique. '

Doterajší stav technikyPrior art

Po tom, čo Merril so spolupracovníkmi publikoval metodu stanovenia bielkovín striebrom, použitím roztoku amoniakálneho strieborného komplexu (Switzer, R.C. , Merril, C.R., Shifrin, S.: Anal. Biochem., 98, 231-237 /1979/) a roztokom dusičnanu strieborného (Merril, C.R., Dunau, M.L., Goldman, D.: Anal. Biochem., 110, 201-207 /1981/) vznikol celý rad róznych metodik, založených na jednom či druhom principe. Tieto metodiky sa vyznačovali postupným zjednodušováním pracovného postupu alebo změnami v predchádzajúcich postupoch, za účelom dosiahnutia lepších výsledkov. Týkalo sa to najma spósobu fixovania bielkovín v géli, spósobu odstraňovania kyseliny octovéj z prostredia před pósobením roztoku striebra, spósobu odstraňovania strieborných solí z gélu, spósobu vyvolávania a spósobu zastavenia vyvolávacieho procesu, avšak i procesu celkového scitlivenia metody. Z velkého množstva róznych metodik sú tu uvedené tie, ktoré sa dodnes najviac používajú a tie, ktoré v niektorom z pracovného postupu priniesli pozoruhodné zlepšenie.After Merril and colleagues published a method for the determination of proteins by silver, using a solution of ammoniacal silver complex (Switzer, RC, Merril, CR, Shifrin, S .: Anal. Biochem., 98, 231-237 (1979)) and a solution of silver nitrate. (Merril, CR, Dunau, ML, Goldman, D .: Anal. Biochem., 110, 201-207 (1981)), a number of different methodologies have been developed, based on one or the other principle. These methodologies were characterized by a gradual simplification of the workflow or changes in previous procedures in order to achieve better results. This concerned in particular the method of fixing the proteins in the gel, the method of removing acetic acid from the medium before the silver solution was treated, the method of removing silver salts from the gel, the method of development and the method of stopping the development process. Of the large number of different methodologies, those that are the most widely used today and those that have brought remarkable improvements in some of the workflows are listed here.

Oakley a spol. (Anal. Biochem., 105, 361-363 /1980/) ako prví použili vodný 10% roztok glutaraldehydu za účelom fixovania bielkovín. To však spósobuje silné žltošedé pozadie. Wray a spol. (Anal. Biochem., 118, 197-203 /1981/) používajú amoniákálny strieborný komplex; podlá ich metodického postupu však gély majú šedé pozadie. Podobné poměrně silné šedé pozadie majú gély farbené striebrom metodou podia Morrisseya a spol. (Anal. Biochem. 117, 307-310 /1981/), ktorí před pósobením 0,1% roztoku dusičnanu strieborného použili roztok ditiotreitolu. Sammons a spol. (Elektrophoresis 2, 135-141 /1981/) publikovali taký metodický postup, pri ktorom sú bielkoviny rožne zafarbené. Gél má však velmi intenzívně oranžové pozadie. Ohsawa a Ebata (Anal. Biochem. 135, 409-415 /1983/) vyvinuli metodu, pri ktorej použitím 70% vodného roztoku polyetylénglykolu 2000 odvodnili gél po elektroforéze a tým odstránili interferujúce látky a súčasne velmi účinné fixovali bielkoviny. Pri reprodukovaní tejto metody vzniklo však také intenzívně šedočierne pozadie, že zóny bielkovín prakticky nebolo možné identifikovat. Biel a spol. (Elektrophoresis, 7, 232-235 /1986/) odporúčajú urýchliů proces farbenia zvýšením teploty roztokov na 60 až 70 °C. Blum a spol. (Elektrophoresis, 8, 93-99 /1987/) používajú redukčně činidlá před pósobením roztoku dusičnanu strieborného na zvýšenie kontrastu a zvýšenie citlivosti metody. Marcinka (CS autorské osvedčenie 259 363) použil fixačno-vymývací roztok s obsahom 0,25 % hmot, glutaraldehydu na zvýšenie citlivosti stanovenia a po roztoku amoniakálneho strieborného komplexu použil poměrně dlhé pranie gélu 0,001 M NaOH na odmytie přebytečných volných iónov striebra z gélu. Tým dosiahol pri vysokej citlivosti stanovenia minimálně pozadie. Všetky opisované metody, ktoré sa zakladajú na použití amoniakálneho strieborného komplexu majú nevýhodu v tom, že sú spojené s vyššou spotřebou striebra a roztok tohoto strieborného komplexu je potenciálně výbušný.Oakley et al. (Anal. Biochem., 105, 361-363 (1980)) were the first to use an aqueous 10% solution of glutaraldehyde to fix proteins. However, this causes a strong yellow-gray background. Wray et al. (Anal. Biochem., 118, 197-203 (1981)) use an ammoniacal silver complex; however, according to their methodological procedure, the gels have a gray background. Gels stained with silver by the method of Morrissey et al. (Anal. Biochem. 117, 307-310 (1981)), who used a solution of dithiothreitol before treatment with a 0.1% solution of silver nitrate. Sammons et al. (Elektrophoresis 2, 135-141 (1981)) have published a method in which proteins are spit-colored. However, the gel has a very intense orange background. Ohsawa and Ebata (Anal. Biochem. 135, 409-415 (1983)) developed a method in which, using a 70% aqueous solution of polyethylene glycol 2000, they dehydrated the gel after electrophoresis, thereby removing interfering substances and at the same time fixing proteins very effectively. However, the reproduction of this method produced such an intense gray-black background that it was practically impossible to identify protein zones. Biel et al. (Elektrophoresis, 7, 232-235 (1986)) recommend accelerating the staining process by raising the temperature of the solutions to 60-70 ° C. Blum et al. (Elektrophoresis, 8, 93-99 (1987)) use reducing agents before exposure to the silver nitrate solution to increase the contrast and increase the sensitivity of the method. Marcinka (CS author's certificate 259 363) used a fixative-elution solution containing 0.25% by weight, glutaraldehyde to increase the sensitivity of the assay and after a solution of ammonia silver complex used a relatively long wash of 0.001 M NaOH gel to wash excess free silver ions from the gel. The team achieved at least background with high sensitivity of the determination. All the methods described, which are based on the use of an ammoniacal silver complex, have the disadvantage that they are associated with a higher consumption of silver and the solution of this silver complex is potentially explosive.

Podstata vynálezu ’SUMMARY OF THE INVENTION

Uvedené nevýhody doterajších metod stanovenia bielkovín detekčnou technikou striebrom, po elektroforetickej frakcionácii bielkovín, praním plátnového gélu po elektroforéze po dobu min. 45 minut fixačno-vymývacím roztokem, odstraňuje spósob stanovenia bielkovín podlá vynálezu. Podstata vynálezu spočívá v tom, že po vypraní fixačno-vymývacím roztokom sa platňový gél premýva aspoň jedenkrát 25 až 50% metanolom alebo etanolom, aspoň jedenkrát vodou a pósobí sa naň roztokom tiosíranu sodného. Potom sa aspoň jedenkrát oplákne vodou a minimálně 15 minut sa naň pósobí farbiacim roztokom obsahujúcim dusičnan strieborný, formaldehyd a například kyselinu citrónovú vo vodě, po ktorom sa premýva vodou jeden až trikrát po dobu 30 až 90 sekúnd a nakoniec sa pósobí vývojkou obsahujúcou vodný roztok formaldehydu, uhličitanu sodného a tiosíranu sodného.The above-mentioned disadvantages of the existing methods for the determination of proteins by the silver detection technique, after electrophoretic fractionation of proteins, by washing the canvas gel after electrophoresis for a period of min. 45 minutes with fixative-elution solution, removes the method for the determination of proteins according to the invention. The invention is based on the fact that, after washing with the fixative-washing solution, the plate gel is washed at least once with 25 to 50% methanol or ethanol, at least once with water and treated with sodium thiosulphate solution. It is then rinsed at least once with water and treated for at least 15 minutes with a coloring solution containing silver nitrate, formaldehyde and, for example, citric acid in water, washed with water one to three times for 30 to 90 seconds and finally treated with a developer containing aqueous formaldehyde. , sodium carbonate and sodium thiosulphate.

Fixačno-vymývací roztok pozostáva zo zmesi metanolu alebo etanolu, kyseliny octovéj a vody v objemových pomeroch 30 až 50 : 12 : 38 a obsahuje formaldehyd v koncentrácii 0,018 až 0,037 % hmot.The fixative-washing solution consists of a mixture of methanol or ethanol, acetic acid and water in volume ratios of 30 to 50: 12: 38 and contains formaldehyde in a concentration of 0.018 to 0.037% by weight.

Roztok tiosíranu sodného sa móže použit v koncentrácii 0,8 mM, ako farbiaci roztok sa móže použit dusičnan strieborný o koncentrácii 0,2 % hmot, a formaldehyd o koncentrácii 0,018 % hmot., ktorý móže naviac obsahovat kyselinu citrónovú v koncentrácii 0,02 % hmot. ·The sodium thiosulphate solution may be used at a concentration of 0.8 mM, silver nitrate at a concentration of 0.2% by weight, and formaldehyde at a concentration of 0.018% by weight, which may additionally contain citric acid at a concentration of 0.02%, may be used as the staining solution. mass ·

Po farbiacom roztoku sa gél vymývá vodou jedenkrát 1 min. a jedenkrát 1/2 min. Zistilo sa tiež, že je výhodné, ked vyvolávací roztok obsahuje formaldehyd o koncentrácii 0,018 % hmot., uhličitan sodný o koncentrácii 5 až 6 % hmot, a tiosíran sodný o koncentrácii 16 μΜ.After the staining solution, the gel is washed once with water for 1 minute. and once 1/2 min. It has also been found to be advantageous if the developing solution contains formaldehyde at a concentration of 0.018% by weight, sodium carbonate at a concentration of 5 to 6% by weight, and sodium thiosulphate at a concentration of 16 μΜ.

Proces vyvolávania sa zastaví pósobením zastavovacieho kúpela obsahujúceho kyselinu citrónovú o koncentrácii 0,1 % hmot, a metanol alebo etanol o koncentrácii 20 % obj. vo vodě a následné sa perie vodou.The development process is stopped by the action of a stopping bath containing citric acid at a concentration of 0.1% by weight, and methanol or ethanol at a concentration of 20% by volume in water, and then washed with water.

Výhoda uvedeného spósobu stanovenia bielkovín spočívá v tom, že vo fixačno-vymývacom roztoku sa namiesto bifunkčného aldehydu (glutaraldehydu) používá účinnější monofunkčný formaldehyd v rádovo nižšej koncentrácii, dalej že sa před pósobením roztoku striebornej soli pósobí na gél roztokom tiosíranu sodného ako redukčného činidla, čím sa zvýši citlivost stanovenia a ostrost zón. Ako strieborná sol sa použije roztok dusičnanu strieborného, ktorý je účinnější ako amoníakálny strieborný komplex, preto sa použije v nižšej koncentrácii, čím sa zníži spotřeba striebra. Vývojka, ktorá obsahuje velmi nízku koncentráciu tiosíranu sodného, rozpúšta soli striebra v géli a tým bráni vzniku pozadia, preto sa skracuje pranie gélu po opracovaní roztokom striebornej soli na jeden a pol minúty.The advantage of this method for determining proteins is that instead of bifunctional aldehyde (glutaraldehyde) a more effective monofunctional formaldehyde is used in the fixation-elution solution in an order of magnitude lower concentration. the sensitivity of the determination and the sharpness of the zones are increased. A silver nitrate solution is used as the silver salt, which is more effective than the ammoniacal silver complex, so it is used in a lower concentration, thereby reducing silver consumption. The developer, which contains a very low concentration of sodium thiosulphate, dissolves the silver salt in the gel and thus prevents the formation of a background, therefore the washing of the gel after treatment with the silver salt solution is shortened to one and a half minutes.

Příklady uskutočnenia vynálezuExamples of embodiments of the invention

Příklad 1Example 1

Po skončení elektroforézy bielkovín v polyakrylamidovom géle hrůbky 1 mni a plochy 150 cm² sa tento gél vymývá 1 h v 200 ml fixačno-vymývacieho roztoku, pozostávajúceho zo zmesi metanol, kyselina octová a voda v objemovom pomere 50 : 12 :· 38, ktorý obsahoval 0,018 % hmot. formaldehydu (0,5 ml 37% roztoku formaldehydu/1 liter), potom sa premyje jedenkrát 15 min v 100 ml 30% metanolu a dvakrát 15 min 250 ml vody, d’alej 1 min v 100 ml 0,0 mM tiosíranu sodného a oplákne sa trikrát 150 ml vody, na gél sa potom pósobí 20 min 100 ml farbiaceho roztoku pozostávajúceho z 0,2 % hmot.' dusičnanu strieborného, 0,018 % hmot, formaldehydu a 0,02 % hmot, kyseliny citrónovéj, gél sa potom premyje jedenkrát 1 min a jedenkrát pol min 250 ml vody a následuje vyvolávanie v 200 ml roztoku obsahujúcom 0,018 % hmot, formaldehydu, 6 % hmot, uhličitanu sodného a 16 μΜ tiosíranu sodného. Po vizualizácii zón sa proces vyvolávania zastaví 10 minútovým praním v 200 ml roztoku obsahujúcom 20 %. obj. metanolu a 0,1 % hmot, kyseliny citrónovéj a nakoniec sa perie 1 hodinu v 300 ml vody pri jednej výměně vody za čerstvú.After completion of protein electrophoresis in a polyacrylamide gel with a thickness of 1 mni and an area of 150 cm ^2, this gel is eluted for 1 h in 200 ml of a fixation-washing solution consisting of a mixture of methanol, acetic acid and water in a volume ratio of 50: 12: · 38 % wt. formaldehyde (0.5 ml of a 37% formaldehyde solution / 1 liter), then washed once for 15 minutes in 100 ml of 30% methanol and twice for 15 minutes with 250 ml of water, then for 1 minute in 100 ml of 0.0 mM sodium thiosulphate and it is rinsed three times with 150 ml of water, then the gel is treated for 20 minutes with 100 ml of a dyeing solution consisting of 0.2% by weight. silver nitrate, 0.018% by weight, formaldehyde and 0.02% by weight, citric acid, the gel is then washed once for 1 minute and once for half a minute with 250 ml of water, followed by development in 200 ml of a solution containing 0.018% by weight of formaldehyde, 6% by weight, sodium carbonate and 16 μΜ sodium thiosulphate. After visualization of the zones, the development process is stopped by washing for 10 minutes in 200 ml of a solution containing 20%. vol. of methanol and 0.1% by weight of citric acid and finally washed for 1 hour in 300 ml of water with one exchange of fresh water.

Příklad 2Example 2

Postupovalo sa ako v příklade 1 s tým rozdielom, že fixačno-vymývací roztok obsahoval 0,037 % hmot, formaldehydu (1,0 ml 37% roztoku formaldehydu/1 liter), vývojka obsahovala 5 % hmot, uhličitanu sodného. Vyvolávanie sa zastavilo praním dvakrát 5 min v 20% metanole obsahujúcom 0,05 % hmot, kyseliny citrónovej a posledně pranie vodou trvalo 40 minút v 300 ml vody pri jednej výměně vody za čerstvú.The procedure was as in Example 1, except that the fixative-washing solution contained 0.037% by weight of formaldehyde (1.0 ml of a 37% formaldehyde solution / 1 liter), and the developer contained 5% by weight of sodium carbonate. The development was stopped by washing twice for 5 minutes in 20% methanol containing 0.05% by weight of citric acid, and the last washing with water lasted 40 minutes in 300 ml of water with one change of fresh water.

V obidvoch príkladoch je možné namiesto metanolu použit etanol v rovnakých koncentráciách.In both examples, ethanol in the same concentrations can be used instead of methanol.

Priemvselná využitelnostIndustrial usability

Metodu je možné využit na stanovenie malých množstiev bielkovín v géli a to najma v biochemických laboratóriách.The method can be used to determine small amounts of protein in a gel, especially in biochemical laboratories.

Claims

1. Method for determination of proteins by detection technique by silver after electrophoretic fractionation of proteins, by washing the plate gel after electrophoresis for min. 45 minutes with fixative-wash solution, characterized in that after washing with the fixative-wash solution, the linen gel is washed at least once with 25 to 50% methanol or ethanol, at least once with water and treated with sodium thiosulphate solution, then rinsed at least once with water and it is treated for at least 15 minutes with a coloring solution containing silver nitrate, formaldehyde and, for example, citric acid in water, after which it is washed with water one to three times for 30 to 90 seconds and finally with a developer containing aqueous formaldehyde, sodium carbonate and sodium thiosulphate.

2. The method for the determination of proteins according to item 1, characterized in that it is washed with a fixative-washing solution consisting of a mixture of methanol or ethanol, acetic acid and water in volume ratios of 30 to 50:12:38 and containing formaldehyde in a concentration of 0.018 to 0.037%. mass

3. The method for the determination of proteins according to item 1, characterized in that the sodium thiosulphate solution is used at a concentration of 0.8 mM.

4. The method for the determination of proteins according to item 1, characterized in that silver nitrate of con. Is used as the staining solution. concentration of 0.2 wt.%, and formaldehyde at a concentration of 0.018 wt. .

5. The method for determining proteins according to items 1 and 4, characterized in that the staining solution additionally contains citric acid in a concentration of 0.02% by weight.

6. The method for determining proteins according to claim 1, characterized in that after the staining solution, the gel is washed with water once for 1 minute and once for half a minute.

7. The method for determining proteins according to item 1, characterized in that the developing solution contains formaldehyde at a concentration of 0.018% by weight, sodium carbonate at a concentration of 5 to 6% by weight, and sodium thiosulfate at a concentration of 16 μΜ.

8. The method for determining proteins according to item 1, characterized in that the development process is stopped by the action of a stopping bath containing citric acid at a concentration of 0.1% by weight and methanol or ethanol at a concentration of 20% by volume in water and then washed with water.