natgillin/translations-raw huggingface dataset:
Use freshly prepared solutions, because CMP degrades in an 8 % trichloroacetic acid environment at room temperature.
R = odzivni faktor vrha CMPA (9.1.1)
At 4 °C the solution remains stable for 24 hours.
H(E) = višina vrha CMPA iz vzorca (E)
In the case of long series of analyses the use of a cooled sample tray in the automatic injector is desirable
Če je W(E) večji od 1 % in razlika med retenzijskim časom pri vzorcu in standardnem vzorcu [5] manjša od 0,2 minute, potem je trdna snov sladke sirotke prisotna.
Note: 8.4.2. may be omitted if the % B at initial conditions is known from previous analyses.
Ni določena.
The chromatogram of the reference sample [5] should be analogous to Figure.
Linearnost
In this figure the CMPA peak is preceded by two small peaks.
Linearno zvezo s korelacijskim koeficientom > 0,99 je treba dobiti v območju od 0 do 16 % sladke sirotke.
It is essential to obtain a similar separation.
Meja 1 % vključuje negotovost zaradi obnovljivosti.
Prior to chromatographic determination of the samples inject 100 μl of the standard sample without rennet whey [0] (5.4.1)
Ni – standard 4.6
The chromatogram should not show a peak at the retention time of the CMPA peak.
Absorbanca (220 nm)
Determine the response factors R by injecting the same volume of filtrate (8.5.1) as used for the samples.
čas (v minutah)
Calculation of the response factor R:
Značilnosti natančnosti analitskih metod.
CMPA peak: R = W/H
Prikaz postopka medlaboratorijske študije.“
R = the response factor of the CMPA peak
Dodajo se naslednje priloge:
H = the height of the CMPA peak
PRILOGA VI
W = the quantity of whey in the standard sample [5].
Metode za analizo masla za zasebno skladiščenje
Calculation of the percentage of rennet whey powder in the sample
Nemastna trdna snov (razen soli)
W(E) = the percentage (m/m) of rennet whey in the sample (E).
Sol
H(E) = the height of the CMPA peak of the sample (E)
PRILOGA VII
If W(E) is greater than 1 % and the difference between the retention time and that of the standard sample [5] is smaller than 0,2 minutes then rennet whey solids are present.
Metode za analizo posnetega mleka v prahu za zasebno skladiščenje
Not determined.
PRILOGA VIII
Linearity
Metode za analizo sira za zasebno skladiščenje
From 0 to 16 % of rennet whey a linear relationship should be obtained with a coefficient of correlation > 0,99.
Za zagotovitev, da sir, izdelan izključno iz ovčjega, kozjega ali bivoličjega mleka ali iz mešanice ovčjega, kozjega in bivoličjega mleka, dejansko ne vsebuje kazeina kravjega mleka, se uporablja analitska metoda iz dodatka.
The 1 % limit includes the uncertainty due to reproducibility.
Če je vsebnost kazeina kravjega mleka v analiziranem vzorcu enaka ali višja od vsebnosti v referenčnem vzorcu, ki vsebuje 1 % kravjega mleka, kakor je opisano v dodatku, se šteje, da je kazein kravjega mleka prisoten.
Ni—4.6 standard
Metode za zaznavanje kazeina kravjega mleka v siru, kakor je navedeno v odstavku 1, se lahko uporabijo, če:
Absorbance (220 nm)
je meja zaznavnosti največ 0,5 % in
time (Minutes)
ni napačnih pozitivnih rezultatov ter
International IDF Standard 135B/1991.
se kazein kravjega mleka lahko zazna z zahtevano občutljivostjo tudi po daljših obdobjih zorenja, do česar lahko pride v običajnih trgovinskih pogojih.
Milk and milk products.
Če katera od navedenih zahtev ni izpolnjena, se uporabijo referenčne metode iz dodatka.
Precision characteristics of analytical methods.
Dodatek
Outline of collaborative study procedure.’
METODA ZA ZAZNAVANJE KRAVJEGA MLEKA IN KAZEINATA V SIRIH IZ OVČJEGA, KOZJEGA ALI BIVOLIČJEGA MLEKA ALI MEŠANIC OVČJEGA, KOZJEGA IN BIVOLIČJEGA MLEKA
the following Annexes are added:
NAMEN
ANNEX VI
Zaznavanje kravjega mleka in kazeinata v sirih iz ovčjega, kozjega in bivoličjega mleka ali mešanic ovčjega, kozjega in bivoličjega mleka z izoelektričnim fokusiranjem γ-kazeinov po plazminolizi.
Methods of analysis of butter under private storage
PODROČJE UPORABE
Non Fat Solids (excluding salt)
Ta metoda je primerna za občutljivo, specifično zaznavanje surovega in toplotno obdelanega kravjega mleka in kazeinata v svežih in zorenih sirih iz ovčjega, kozjega in bivoličjega mleka ter iz mešanic ovčjega, kozjega in bivoličjega mleka.
Salt
Ni pa primerna za zaznavanje mlečnih ali sirnih ponaredkov s toplotno obdelanimi proteinskimi koncentrati govejega sirila.
The method to be applied shall be approved by the paying agency.
Izolacija kazeinov iz sira in referenčni standardi
ANNEX VII
Raztopitev izoliranih kazeinov in izpostavitev v plazminsko cepitev (EC.3.4.21.7)
Methods of analysis of skimmed milk powder under private storage
Izoelektrično fokusiranje plazminsko obdelanih kazeinov v prisotnosti sečnine in obarvanje proteinov
ANNEX VIII
Ocenjevanje obarvanih γ3 in γ2-kazeinskih profilov (dokaz kravjega mleka) s primerjavo primerka, pridobljenega iz vzorca, s primerki, pridobljenimi v istem gelu iz referenčnih standardov, ki vsebujejo 0 % in 1 % kravjega mleka.
Methods of analysis of cheeses under private storage
Razen če ni določeno drugače, se uporabijo analitsko čiste kemikalije.
The method of analysis laid down in the Appendix shall be used to ensure that cheese made exclusively from ewe's milk, goat's milk or buffalo milk or from a mixture of ewe's milk, goat's milk and buffalo milk does not contain cow's milk casein.
Voda je dvakrat destilirana ali enakovredne čistosti.
Cow's milk casein is considered to be present if the cow's milk casein content of the analysed sample is equal to or higher than the content of the reference sample containing 1 % cow's milk as laid down in the Appendix.
Opomba: Naslednje podrobnosti veljajo za laboratorijsko pripravljene poliakrilamidne gele v velikosti 265 × 125 × 0,25 mm, ki vsebujejo sečnino.
Methods for detecting cow's milk casein in cheeses referred to in paragraph 1 may be used provided that:
Če so uporabljene druge velikosti in vrste gelov, se lahko pogoji ločitve prilagodijo.
the detection limit is maximum 0,5 % and
Izoelektrično fokusiranje
there are no false-positive results and
Reagenti za izdelavo poliakrilamidnih gelov z vsebnostjo sečnine
cow's milk casein is detectable with the required sensitivity even after long ripening periods, as may occur in usual commercial conditions.
Osnovna gelska raztopina
If any of the above mentioned requirements is not met, the methods laid down in the Appendix shall be used.
V vodi raztopite:
Appendix
4,85 g akrilamida,
METHOD FOR THE DETECTION OF COW'S MILK AND CASEINATE IN CHEESES FROM EWE'S MILK, GOAT'S MILK OR BUFFALO MILK OR MIXTURES OF EWE'S MILK, GOAT'S MILK AND BUFFALO MILK
0,15 g N, N′-metilen-bis-akrilamida (BIS),
SCOPE
48,05 g sečnine,
Detection of cow's milk and caseinate in cheeses made from ewe's milk, goat's milk, buffalo milk or mixtures of ewe's, goat's and buffalo milk by isoelectric focusing of γ-caseins after plasminolysis.
15,00 g glicerola (87 % w/w)
FIELD OF APPLICATION
in dopolnite do 100 ml ter raztopino shranite v rjavi steklenici v hladilniku.
The method is suitable for sensitive and specific detection of native and heat-treated cow's milk and caseinate in fresh and ripened cheeses made from ewe's milk, goat's milk, buffalo milk or mixtures of ewe's, goat's and buffalo milk.
Opomba: Namesto navedenih določenih količin nevrotoksičnih akrilamidov se lahko uporabi tudi komercialo dostopna mešanica akrilamidne/BIS raztopine.
It is not suitable for the detection of milk and cheese adulteration by heat-treated bovine whey protein concentrates.
Če taka raztopina vsebuje 30 % w/v akrilamida in 0,8 % w/v BIS, se namesto navedenih količin za pripravek vzame 16,2 ml.
Isolation of caseins from cheese and the reference standards
Rok uporabnosti raztopine je največ 10 dni.
Dissolving of the isolated caseins and submitting to plasmin (EC.3.4.21.7) cleavage
Če je njena prevodnost več kot 5 μS, raztopino deionizirajte z mešanjem 2 g amberlita MB-3 30 minut, nato filtrirajte skozi membrano 0,45 μm.
Isoelectric focusing of plasmin-treated caseins in the presence of urea and staining of proteins
Gelska raztopina
Evaluation of stained γ3 and γ2 -casein patterns (evidence of cow's milk) by comparison of the pattern obtained from the sample with those obtained in the same gel from the reference standards containing 0 % and 1 % cow's milk.
Gelsko raztopino pripravite z mešanjem dodatkov, amfolitov (*) in gelske raztopine iz zaloge (glej 4.1.1).
Unless otherwise indicated, analytical grade chemicals shall be used.
9,0 ml osnovne gelske raztopine,
Water shall be double-distilled or of equivalent purity.
24 mg β-alanina,
Note: The following details apply to laboratory prepared polyacrylamide gels containing urea, of dimensions 265 × 125 × 0,25 mm.
500 μl amfolita pH 3,5–9,5,
Where other sizes and types of gel are used, the separation conditions may have to be adjusted.
250 μl amfolita pH 5-7,
Isoelectric focusing
250 μl amfolita pH 6-8.
Reagents for production of the urea containing polyacrylamide gels
Zmešajte gelsko raztopino in jo dve do tri minute razplinjajte v ultrazvočni kopeli ali vakuumu.
Stock gel solution
Opomba: Gelsko raztopino pripravite, tik preden jo nalijete (glej 6.2).
Dissolve:
Katalizatorske raztopine
4,85 g acrylamide
N, N, N′ N′ – tetrametiletilendiamin (Temed)
0,15 g N, N′-methylene-bis-acrylamide (BIS)
40 % w/v amonijev persulfat (PER):
48,05 g urea
Raztopite 800 mg PER v vodi in dopolnite do 2 ml.
15,00 g glycerol (87 % w/w),
Opomba: Vedno uporabite sveže pripravljeno raztopino PER.
in water and make up to 100 ml and store in a brown glass bottle in the refrigerator.
Kontaktna tekočina
Note: A commercially available pre-blended acrylamide/BIS solution may be used in preference to the quoted fixed weights of the neurotoxic acrylamides.
Kerozen ali tekoči parafin.
Where such a solution contains 30 % w/v acrylamide and 0,8 % w/v BIS, a volume of 16,2 ml shall be used for the formulation instead of the fixed weights.
Anodna raztopina
The shelf life of the stock solution is a maximum of 10 days; if its conductivity is more than 5 μS, de-ionize by stirring with 2 g Amberlite MB-3 for 30 minutes, then filter through a 0,45 μm membrane.
Raztopite 5,77 g fosforjeve kisline (85 % w/w) v vodi in dopolnite do 100 ml.
Gel solution
Katodna raztopina
Prepare a gel solution by mixing additives and ampholytes (*) with the stock gel solution (see 4.1.1).
Raztopite 2,00 g natrijevega hidroksida v vodi in dopolnite do 100 ml.
9,0 ml stock solution
Priprava vzorca
24 mg β-alanine
Reagent za izolacijo proteinov
500 μl ampholyte pH 3,5-9,5
Razredčena ocetna kislina (25,0 ml ledocetne kisline dopolnite z vodo do 100 ml).
250 μl ampholyte pH 5-7
Diklorometan
250 μl ampholyte pH 6-8
Aceton
Mix the gel solution and de-gas for two to three minutes in an ultrasonic bath or in vacuum.
Pufer za raztapljanje proteinov
Note: Prepare the gel solution immediately prior to pouring it (see 6.2).
V vodi raztopite
Catalyst solutions
5,75 g glicerola (87 % w/w),
N, N, N′ N′ — tetramethylethylenediamine (Temed)
24,03 g sečnine,
40 % w/v ammonium persulphate (PER):
250 mg ditiotreitola
Dissolve 800 mg PER in water and make up to 2 ml.
in dopolnite do 50 ml.
Note: Always use freshly prepared PER solution.
Opomba: Hranite v hladilniku; najdaljši rok uporabnosti je en teden.
Contact fluid
Reagenti za plazminsko cepitev kazeinov
Kerosene or liquid paraffin
Pufer amonijevega hidrogenkarbonata
Anode solution
Titrirajte 0,2 mol/l raztopine amonijevega hidrogenkarbonata (1,58 g/100 ml vode), ki vsebuje 0,05 mol/l etilendiamintetraocetne kisline (EDTA, 1,46 g/100 ml) z 0,2 mol/l raztopine amonijevega karbonata (1,92 g/100 ml vode), ki vsebuje 0,05 mol/l EDTA do pH 8.
Dissolve 5,77 g phosphoric acid (85 % w/w) in water and dilute to 100 ml.
Goveji plazmin (EC.
Cathode solution
3.4.21.7) z aktivnostjo najmanj 5 U/ml.
Dissolve 2,00 g sodium hydroxide in water and dilute to 100 ml with water.
Raztopina ε-aminokapronske kisline za inhibiranje encimov.
Sample preparation
Raztopite 2,624 g ε-aminokapronske kisline (6-amino-n-heksanojske kisline) v 100 ml 40 % (v/v) etanola.
Reagents for protein isolation
Standardi
Dilute acetic acid (25,0 ml of glacial acetic acid made up to 100 ml with water)
Certificirani referenčni standardi mešanice sesirjenega posnetega ovčjega in kozjega mleka, ki vsebuje 0 % in 1 % kravjega mleka, so na voljo na Inštitutu za referenčne materiale in meritve Komisije, 2440 Geel, Belgija.
Dichloromethane
Priprava laboratorijskih delovnih standardov sesirjenega bivoličjega mleka, ki vsebuje 0 % in 1 % kravjega mleka.
Acetone
Posneto mleko se pripravi z 20-minutnim centrifugiranjem vzorca surovega bivoličjega mleka ali mleka goveda pri 37 °C in 2500 g.
Protein dissolving buffer
Po hitri ohladitvi epruvete in vsebine na 6 do 8 °C se popolnoma odstrani zgornja plast maščobe.
Dissolve
Za pripravo 1 % standarda v litrski čaši k 495 ml posnetega bivoličjega mleka dodajte 5,00 ml posnetega mleka goveda, uravnajte pH na 6,4 z dodajanjem razredčene mlečne kisline (10 % w/v).
5,75 g glycerol (87 % w/w)
Nastavite temperaturo na 35 °C in dodajte 100 μl telečjega sirila (aktivnost sirila je 1: 10000, pribl.
24,03 g urea
3000 U/ml), mešajte eno minuto in nato čašo pustite eno uro pokrito z aluminijasto folijo pri 35 °C, da se naredi sirna gruda.
250 mg dithiothreitol,
Ko se je naredila sirna gruda, se celotno sesirjeno mleko posuši z zmrzovanjem (freeze-dry) brez predhodne homogenizacije ali odcejanja sirotke.
in water and make up to 50 ml
Po tem postopku sirno grudo fino zmeljite, da dobite homogen prah.
Note: Store in a refrigerator, maximum shelf-life one week.
Za pripravo 0 % standarda uporabite isti postopek z naravnim posnetim bivoličjim mlekom.
Reagents for plasmin cleavage of caseins
Standard se shrani pri – 20 °C.
Ammonium carbonate buffer
Opomba: Pred pripravo standardov je priporočljivo, da se preveri čistost bivoličjega mleka z izoelektričnim fokusiranjem plazminsko obdelanih kazeinov.
Titrate a 0,2 mol/l ammonium hydrogencarbonate solution (1,58 g/100 ml water) containing 0,05 mol/l ethylenediaminetetraacetic acid (EDTA, 1,46 g/100 ml with a 0,2 mol/l ammonium carbonate solution (1,92 g/100 ml water) containing 0,05 mol/l EDTA to pH 8.
Reagenti za obarvanje proteinov
Bovine plasmin (EC.
Fiksir
3.4.21.7), activity at least 5 U/ml
Raztopite 150 g trikloroocetne kisline v vodi in dopolnite do 1000 ml.
ε-Aminocaproic acid solution for enzyme inhibition
Raztopina za razbarvanje
Dissolve 2,624 g ε-aminocaproic acid (6 amino-n-hexanoic acid) in 100 ml of 40 % (v/v) ethanol.
500 ml metanola in 200 ml ledocetne kisline razredčite z destilirano vodo do 2000 ml.
Standards
Opomba: Vsak dan pripravite svežo raztopino za razbarvanje.
Certified reference standards of a mixture of renneted ewe's and goat's skimmed milk containing 0 % and 1 % of cow's milk are available from the Commission's Institute for Reference Materials and Measurements, B-2440 Geel, Belgium
Lahko jo pripravite z mešanjem enakih prostornin osnovnih raztopin 50 % (v/v) metanola in 20 % (v/v) ocetne kisline.
Preparation of laboratory interim-standards of buffalo's renneted milk containing 0 % and 1 % of cow's milk
Raztopine za obarvanje
Skimmed milk is prepared by centrifuging of either buffalo or bovine raw bulk milk at 37 °C at 2500 g for 20 minutes.
Raztopina za obarvanje (osnovna raztopina 1)
After cooling the tube and contents rapidly to 6 to 8 °C, the upper fat layer is removed completely.
Raztopite 3,0 g Coomassie Brillant Blue G-250 (C.I. 42655) v 1000 ml 90 % (v/v) metanola z magnetnim mešalom (približno 45 minut), filtrirajte skozi dva srednje hitra nagubana filtra.
For the preparation of the 1 % standard add 5,00 ml of bovine skimmed milk to a 495 ml of buffalo's skimmed milk in a 1 l beaker, adjust the pH to 6,4 by the addition of dilute lactic acid (10 % w/v).
Raztopina za obarvanje (osnovna raztopina 2)
Adjust the temperature to 35 °C and add 100 μl of calf rennet (rennet activity 1: 10000, c.
Raztopite 5,0 g bakrovega sulfata pentahidrata v 1000 ml 20 % (v/v) ocetne kisline.
3000 U/ml), stir for 1 minute and then leave the beaker covered with an aluminium foil at 35 °C for one hour to allow formation of the curd.
Raztopina za obarvanje (delovna raztopina)
After the curd has formed, the whole renneted milk is freeze-dried without prior homogenization or draining of the whey.
Tik pred obarvanjem zmešajte skupaj 125 ml vsake osnovne raztopine (4.11.1, 4.11.2).
After freeze-drying it is finely ground to produce a homogeneous powder.
Opomba: Raztopino za obarvanje je treba pripraviti na dan uporabe.
For the preparation of the 0 % standard, carry out the same procedure using genuine buffalo skimmed milk.
OPREMA
The standards shall be stored at – 20 °C.
Steklene ploščice (265 × 125 × 4 mm); valjček iz gume (širine 15 cm); nivelirna miza
Note: It is advisable to check the purity of the buffalo milk by isoelectric focusing of the plasmin-treated caseins before preparation of the standards.
Nosilna folija za gel (265 × 125 mm)
Reagents for protein staining
Pokrivna folija (280 × 125 mm).
Fixative
Na vsak dolgi rob zalepite trak lepilnega traku (280 × 6 × 0,25 mm) (glej sliko 1)
Dissolve 150 g trichloroacetic acid in water and make up to 1000 ml.
Elektrofokusirna komora s hladilno ploščo (npr. 265 × 125 mm) in ustrezen vir energije (≥ 2,5 kV) ali avtomatska elektroforezna naprava
Destaining solution
Cirkulacijski kriostat, termostatsko nadzorovan na 12 ± 0,5 °C
Dilute 500 ml methanol and 200 ml glacial acetic acid to 2000 ml with distilled water.
Centrifuga, nastavljiva do 3000 g
Note: Prepare the destaining solution fresh every day; it can be prepared by mixing equal volumes of stock solutions of 50 % (v/v) methanol and 20 % (v/v) glacial acetic acid.
Elektrodni trakovi (≥ 265 mm dolgi)
Staining solutions
Plastične kapalke za anodne in katodne raztopine
Staining solution (stock solution 1)
Trakovi za nanos vzorca (10 × 5 mm, iz viskoze ali nizkoadsorpcijski filtrirni papir)
Dissolve 3,0 g Coomassie Brilliant Blue G-250 (C.I. 42655) in 1000 ml 90 % (v/v) methanol using a magnetic stirrer (approximately 45 minutes), filter through two medium-speed folded filters.
Posode iz nerjavnega jekla ali steklene posode za obarvanje in razbarvanje (npr. pladnji za instrumente 280 × 150 mm)
Staining solution (stock solution 2)
Prilagodljiv palični homogenizator (premer osi 10 mm), v razponu od 8000 do 20000 vrtljajev/minuto
Dissolve 5,0 g copper sulphate pentahydrate in 1000 ml 20 % (v/v) acetic acid.
Varilnik folije
Staining solution (working solution)
Mikropipete s prostornino 25 μl
Mix together 125 ml of each of the stock solutions (4.11.1, 4.11.2) immediately prior to staining.
Vakuumski koncentrator ali – zamrzovalni sušilnik
Note: The staining solution should be prepared on the day that it is used.
Termostatsko nadzorovana vodna kopel, prilagodljiva na 35 in 40 ± 1 °C, z mešalnikom
EQUIPMENT
Denzitometer za odčitavanje pri λ = 634 nm
Glass plates (265 × 125 × 4 mm); rubber roller (width 15 cm); levelling table
Izolacija kazeinov
Gel carrier sheet (265 × 125 mm)
Stehtajte količino, enakovredno 5 g suhe mase sira ali referenčnih standardov v 100-mililitrski epruveti za centrifugiranje, dodajte 60 ml destilirane vode in homogenizirajte s paličnim homogenizatorjem (8000 do 10000 vrt./min).
Covering sheet (280 × 125 mm).
Uravnajte na pH 4,6 z razredčeno ocetno kislino (4.5.1) in centrifugirajte (5 minut, 3000 g).
Stick on strip of adhesive tape (280 × 6 × 0,25 mm) to each long edge (see Figure 1)
Odlijte maščobo in sirotko, homogenizirajte ostanek pri 20000 vrt./min v 40 ml destilirane vode s pH 4,5, dobljenim z razredčeno ocetno kislino (4.5.1); dodajte 20 ml diklorometana (4.5.2), znova homogenizirajte in centrifugirajte (5 minut, 3000 g).
Electrofocusing chamber with cooling plate (e.g. 265 × 125 mm) and suitable power supply (≥ 2,5 kV) or automatic electrophoresis device
Z lopatico odstranite kazeinsko plast, ki leži med vodno in organsko fazo (glej sliko 2), in odlijte obe fazi.
Circulation cryostat, thermostatically controlled at 12 ± 0,5 °C
Znova homogenizirajte kazein v 40 ml destilirane vode (glej zgoraj) in 20 ml diklorometana (4.5.2) in centrifugirajte.
Centrifuge, adjustable to 3000 g
Nadaljujte postopek (dva- do trikrat), dokler obe ekstrakcijski fazi ne postaneta brezbarvni.
Electrode strips (≥ 265 mm long)
Homogenizirajte proteinski preostanek s 50 ml acetona (4.5.3) in filtrirajte prek srednje hitrega nagubanega filtrirnega papirja.
Plastic dropping bottles for the anode and cathode solutions
Ostanek na filtru vsakič sperite z dvema ločenima 25-mililitrskima odmerkoma acetona in pustite, da se posuši na zraku ali v toku dušika, nato ga v terilnici zdrobite v fin prah.
Sample applicators (10 × 5 mm, viscose or low protein-adsorption filter paper)
Opomba: Izoliran suh kazein je treba hraniti pri – 20 °C.
Stainless steel or glass staining and destaining dishes (e.g. 280 × 150 mm instrument trays)
Plazminsko cepljenje β-kazeinov za okrepitev γ-kazeinov
Adjustable rod homogenizer (10 mm shaft diameter), rpm range 8000 to 20000
Disperzirajte 25 mg izoliranih kazeinov (6.1.1) v 0,5 ml pufra amonijevega karbonata (4.7.1) in 20 minut homogenizirajte, npr. z uporabo ultrazvočnega postopka.
Film welder
Ogrejte na 40 °C in dodajte 10 μl plazmina (4.7.2), zmešajte in eno uro inkubirajte pri 40 °C z neprestanim tresenjem.
25 μl micropipettes
Za inhibicijo encima dodajte 20 μl raztopine ε-aminokapronske kisline (4.7.3), nato dodajte 200 mg sečnine v trdi obliki in 2 ml ditiotreitola.
Vacuum concentrator or freeze-dryer
Opomba: Da bi dobili več simetrije v fokusiranih kazeinskih trakovih, je priporočljivo, da raztopino sušite z zmrzovanjem, potem ko ste dodali ε-aminokapronsko kislino, in nato raztopite preostanek v 0,5 ml pufra za raztapljanje proteinov (4.6).
Thermostatically controlled water bath adjustable to 35 and 40 ± 1 °C with shaker
Priprava sečnine, ki vsebuje poliakrilamidne gele
Densitometer equipment reading at λ = 634 nm
Z nekaj kapljicami vode povaljajte nosilno folijo za gel (5.2) po stekleni plošči (5.1), odstranite vso odvečno vodo s papirnato brisačo ali tkanino.
Isolation of caseins
Na enak način povaljajte pokrivno folijo (5.3) z distančniki (0,25 mm) po drugi stekleni plošči.
Weigh the amount equivalent to 5 g dry mass of cheese or the reference standards into a 100 ml centrifuge tube, add 60 ml distilled water and homogenize with a rod homogenizer (8000 to 10000 rpm).
Položite ploščo vodoravno na nivelirno mizo.
Adjust to pH 4,6 with dil. acetic acid (4.5.1) and centrifuge (5 minutes, 3000 g).
Dodajte 10 μl Temeda (4.1.3.1) v pripravljeno in razplinjeno gelsko raztopino (4.1.2), pomešajte in dodajte 10 μl raztopine PER (4.1.3.2), dobro premešajte in takoj enakomerno nalijte na sredino pokrivne folije.
Decant the fat and whey, homogenize the residue at 20000 rpm in 40 ml distilled water adjusted to pH 4,5 with dil. acetic acid (4.5.1), add 20 ml dichloromethane (4.5.2), homogenize again and centrifuge (5 minutes, 3000 g).
En rob nosilne plošče gela (folija naj bo obrnjena navzdol) položite na pokrivno folijo in jo počasi spuščajte, tako da se med folijami naredi film gela in se enakomerno ter brez mehurčkov razširi (slika 3).
Remove the casein layer that lies between the aqueous and organic phases (see Figure 2) with a spatula and decant off both phases.
Pazljivo do konca spustite nosilno ploščo gela s tanko lopatico in nanjo položite še tri steklene plošče, ki bodo namenjene za uteži.
Rehomogenise the casein in 40 ml distilled water (see above) and 20 ml dichloromethane (4.5.2) and centrifuge.
Potem ko je polimerizacija končana (po približno 60 minutah), premestite polimerizirani gel na nosilno folijo skupaj s pokrivno folijo, tako da rahlo udarite po steklenih ploščah.
Repeat this procedure until both extraction phases are colourless (two to three times).
Skrbno očistite hrbtno stran nosilne folije ostankov gela in sečnine.
Homogenize the protein residue with 50 ml acetone (4.5.3) and filter through a medium-speed folded filter paper.
Zavarite gelov sendvič v filmski tulec in shranite v hladilnik (največ šest tednov).
Wash the residue on the filter with two separate 25 ml portions of acetone each time and allow to dry in the air or a stream of nitrogen, then pulverize finely in a mortar.
Opomba: Pokrivna folija z distančniki se lahko uporabi znova.