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ReAction No.3 Product Selection September 2015

Protease InhibitorsIn all eukaryotic cells and bacteria a large number of proteases are located in various compartments, the cytosol, mitochondria, vacuoles, lysosomes, ER, or in the extracellular space. Intracellular proteases are essential regulators in the synthesis, activation and degradation of proteins. Extracellular or secreted proteases are most prominent in the intestinal

tract of animals or as a part of the blood-clotting cascade. Accordingly, different tissues or organisms contain different sets of proteases. Knowledge of the protease set of a particular expression system enables researchers to combat protease activity throughout the procedure of purifi cation and analysis of proteins.

Description Target Protease Class / Target Enzymes Order No. Quantity

AEBSF Hydrochloride BioChemica serine proteases, thrombin, chymotrypsin, kallikrein, plasmin, proteinase K, trypsin

A1421,0250A1421,0500

250 mg500 mg

Antipain Dihydrochloride BioChemica

serine/cysteine proteases, trypsin, papain, cathepsin B

A2129,0010A2129,0025

10 mg25 mg

Aprotinin BioChemica serine proteases, trypsin, chymotrypsin, kallikrein, plasmin

A2132,0025A2132,0100

25 mg100 mg

Bestatin Hydrochloride BioChemica

amino-peptidase B, leucine amino-peptidase, tripeptide amino-peptidase, aminopeptidases of the cell surface

A2137,0025 25 mg

E-64N-(trans-Epoxy succinyl)- L-leucine-4-guanidinobutylamide

cysteine proteases, papain, bromelain, calpain, cathepsin B, H, L, tumor cathepsin, Streptococcus protease, fi cin

A2157,0005A2157,0010

5 mg10 mg

Leupeptin Hemisulfate serine/cysteine proteases, plasmin, trypsin, papain, cathepsin B, thrombin, calpain

A2183,0025 25 mg

Pepstatin A acid proteases, aspartic proteases, pepsin, cathepsin D, renin, HIV- und MMTV-proteases

A2205,0025 25 mg

Protease Inhibitor Cocktail 6 His-Tag Prot

mixture of inhibitors optimized for the purifi cation of His-Tag proteins from cell extracts. Contains AEBSF, Bestatin, E-64, Pepstatin A, and Phosphoramidon. Ready-to-use solution

A7802,0001 1 ml

2 ReAction No.3 Biochemistry

Biochemistry ReAction No.3 3

Gold AB-HisDetect is a Gold conjugated anti-His-Tag antibody solution. His-tagged proteins are specifi cally stained in pink directly on the blot. The stain gives a quantitative and permanent signal. Stained protein is seen with the naked eye and may be recorded simply with a camera or scanner.

¡ Detection limit within the pico-molar range comparable to ECL.¡ Western blot staining within only 60 minutes ¡ Does not require luminescence detection

A BB

Protease inhibitors often resemble structural elements of the respective protease substrate. They bind more or less specifi cally either reversibly or irreversibly to their target protease. Two examples are A. AEBSF acts by irre-versible inhibition by sulfonylation of a functional group

in the active center of the protease. B. Bestatin resembles a Phe-Leu substrate dipeptide, but the fi rst residue contains an α-hydroxy group resulting in competitive active site-directed inhibition.

How protease inhibitors work

Chemicals structures of (A) AEBSF hydrochloride and (B) Bestatin hydrochloride

Quick check for His-Tag protein expressionRapid detection of over-expressed His-Tag proteins using Gold AB-HisDetect

Description Order No. Quantity

Gold AB-HisDetect A9747,0030 30 ml

4 ReAction No.3 Biochemistry

Novel antibiotics for Cell Culture – an alternative to Pen:Strep

Incubator-Clean™ and Incuwater-Clean™

CellCultureGuard is a combination of novel antibiotics that prevent microbial contaminations in animal and human cell cultures. Providing protection against extra- and intracellular growing bacteria (including mycoplasma), protozoa and fungi (yeast), CellCultureGuard replaces conventional antibiotics such as Penicillin-Streptomycin, Gentamycin or Amphotericin B.

Incubator-Clean™ solution is a spray to prevent contamination of cell culture incubators. The solution prevents growth of fungi, molds, bacteria (and spores), mycoplasma and eliminates many viruses.

Incuwater-Clean™. The water required to create the humidity is a source of contamination which disperses in the incubator.

Application: CellCultureGuard is a 100-fold concentrated sterile solution. Add 1 ml of CellCultureGuard to 100 ml of cell culture medium. CellCultureGuard is stable in cell cultures for 7 days at 37 °C. Store at -20 °C.

Application: Spray CO2 incubators every other week using Incubator-Clean™.

The active ingredients are quaternary benzylammonium compounds. Incubator -Clean™ is non-toxic and biodegradable (it does not contain mercury, formaldehyde, phenol or alcohol).

Application: Change the incubator water with fresh sterile water every two to four weeks, adding 50 ml of Incuwater-Clean™ per 5 liters of water.


CellCultureGuard A8906,0050 50 ml


Incuwater-Clean™100x concentrate solution

A5219,0100 100 ml

Incubator-Clean™ A5230,0500 500 ml

Biochemistry ReAction No.3 5

Related products for cell culture

Get the most out of your transfections

Medium + DNA(or RNA)(or RNA)(or RNA)

Medium + AppliFect LowToxAppliFect LowToxAppliFect LowTox

Incubate cellsTest gene activity after 1–3 days

Add complexto cellsto cells

combinecombine

AppliFect LowTox is an improved transfection reagent for liposome-mediated transfection of mammalian cells.

¡ For highest transfection effi ciency at minimal cytotoxic effects.

A. Outline of the fast Transfection protocol using AppliFect LowTox

B. Overlay of transmitted light and fl uorescence signal of HEK293T cells transfected with an eGFP fusion protein using AppliFect LowTox.


AppliFect LowTox A9027,0001 1 ml

A B


AC-Trypsin – Solution for cell culture A8336,0500 500 ml

Trypsin 1 : 250 from porcine pancreas A4148,0025 25 g

Dimethyl Sulfoxide for cell culture A3672,0100 A3672,0250

100 ml250 ml

L-Glutamine for cell culture A3704,0100A3704,0500

100 g500 g

PBS tablets pH 7.4 (for 100 ml) A9162,0100 100 tabs

PBS tablets pH 7.4 (for 200 ml) A9177,0100 100 tabs

6 ReAction No.3 Microscopy & Histology

Reagents for Clinical DiagnosisHistofi x® Substitute of Formaldehyde

Commission Regulation (EU) 605/2014 and amendment No. 2015/491 establish new rules for classifi cation and labeling of dangerous substances and precautionary statements and use of these substances.

Formaldehyde is, among others, one of the substances affec-ted by this new European legislation, so that we recommend the use of alternative substances and decreasing the exposure times of the users to this product.

PanReac AppliChem has developed a new product that assists to achieve the objectives of the new regulations:

The new Histofi x® Substitute of Formaldehyde is a fi xing agent commonly used in histological techniques, based on glyoxal, which reduces the health hazards for users.

Main advantages:P It is not carcinogenic.P Flexible. It adapts to different fi xation procedures:

product concentrated and ready to useP Convenient and practical packaging (1 and 5 liters)

depending on the needs of each user.P Less pungent odour than formaldehyde.


Histofi x® Substitute of Formaldehyde for clinical diagnosis 255805.2711255805.2714

1000 ml5 L

Histofi x® Substitute of Formaldehyde ready to use for clinical diagnosis 257157.1211257157.1214

1000 ml5 L

Reagents for food analysis ReAction No.3 7

During their handling and processing, milk and dairy products are subjected to stringent analytical controls to guarantee their composition and quality. Commission Regulation (EC) No 273/2008, of 5 March 2008, lays down parameters to determine the reference limits and methods for the chemical, physical and microbiological analysis, and for the organolep-tic evaluation of milk and dairy products.

One of the most important parameters is fat content.Fat content determination is of great importance because:¡ This parameter impacts on the price paid per liter of milk.¡ It is used to determine if a sample of milk or cheese com-

plies with established legal values.plies with established legal values.¡ It is necessary to know its value to classify the milk for the

preparation of derivatives.

Fat content determination in Dairy products

MILK CHEESE

Description Order No. Quantity Gravimetric method(ISO 1211)

Butyrometer method(Gerber method) (ISO 2446)

Gravimetric method(ISO 1735)

Butyrometer method(Van Gulik method) (ISO 3433)

Ammonia 25 % (as NH3) (Reag. USP, Ph. Eur.) for analysis

121129 1000 ml, 2.5 L, 5 L P

Amyl Alcohol according to NF V 04-210 for analysis

125715 1000 mlP

Diethyl Ether stabilized with ~6 ppm of BHT (Reag. Ph. Eur.) for analysis, ACS, ISO

132770 1000 ml, 2.5 L, 5 L P P

Ethanol 96 % v/v for analysis, ACS

131085 1000 ml, 2.5 L, 5 L P P

Hydrochloric Acid 25 % for analysis, ISO

133378 1000 ml, 2.5 L, 5 L P

Isoamyl Alcoholaccording to Gerber for analysis

121079 1000 ml, 2.5 L, 5 L P

Petroleum Ether 40 – 60 °Cfor analysis, ACS, ISO

131315 1000 ml, 2.5 L, 5 L P P

Sulfuric Acid 90 – 91 % according to Gerber for analysis

121010 1000 ml, 2.5 L, 5 L P

Sulfuric Acid 62 % (d= 1.522) according to Van Gulik for analysis

173253 1000 ml, 2.5 L, 5 L P

There are different methods for determining the content of fat in milk and cheese.

Two typical methods are presented:1. Mojonnier method: gravimetric method that uses

organic solvents to extract fat. Subsequently the sol-vent is evaporated and the fat is determined by weig-hing the dry fatty extract.

2. Gerber method: volumetric method that uses chemical reagents (sulfuric acid, detergents) to achieve the breaking of the emulsion and the fat separation. Then the fat content is measured in special flask (butyrometer).

8 ReAction No.3 Reagents for food analysis

Gravimetric method

Butyrometer method

+ 10 g sample (milk) or 1– 3 g sample (cheese)+ 2 ml NH

3 25 % (for milk) or + 8 – 10 ml HCl 25 %

(for cheese) and heat+ 10 ml ethanol+ 2 drops Congo Red+ 25 ml Diethyl Ether+ 25 ml Petroleum Ether+ 25 ml Petroleum Ether

+ 10 ml Sulfuric Acid (90-91 % (d=1.820 g/mL) for milk or 62 % + 10 ml Sulfuric Acid (90-91 % (d=1.820 g/mL) for milk or 62 % (d=1.522 g/mL) for cheese)

+ 11 ml sample (milk) or 3 g sample (cheese)+ 1 ml Isomyl Alcohol for milk or 1 ml Amyl Alcohol for cheese+ 1 ml Isomyl Alcohol for milk or 1 ml Amyl Alcohol for cheese

Close and shake

Close andShake vigorously

Fat collection fl ask (i.e. boiling fl ask)

Distill off the solvent and weigh

CentrifugateLeave in bath (65 – 66 ºC)

approx. 3 min.

Sulfuric acid

Direct read: Fat column from milk or cheese (4.5-1= 3.5 %

of fat)

Aqueous phase

!

Interface

!

Solvent

!

}

Butyrometer DIN12836 for determining the fat content according to Gerber

Fat extraction fl ask(Mojonnier type)

Reagents for food analysis ReAction No.3 9

Kjeldahl Nitrogen determinationProteins are of indispensable nutritional value for humans and animals and are contained in the most common foodstuffs (juices, dairy products, meat, cereals, feed). In fact, the protein content is one of the important parameters declared on nutrition fact labels.

The Kjeldahl method allows the calculation of protein contents in food samples. It is based on the nitrogen determination which is a general constituent of all proteins.

The scope of Kjeldahl nitrogen determinations today also includes applications in the fi elds of environmental analysis, research and development, pharmaceutical, chemicaland cosmetics industries.

The Kjeldahl procedure involves three major steps:

1. Digestion: The sample is mixed with sulfuric acid at temperatures between 340 and 370 ºC. The organic bonded nitrogen is converted into ammonium ions. Organic carbon and hydrogen form carbon dioxide and water. Potassium sulfate and catalysts are added in order to increase the boiling point of sulfuric acid and to decrease the digestion time.

Protein (-Protein (-N) + H) + H22SOSO

44 "" ( (NH

44))

22SOSO

44 + CO + CO

22 + H

22O O

catalyst

2. Distillation: Prior to the distillation the acidic sample is neutralized by means of concentrated sodium hydroxide solution (NaOH).

In the distillation step the ammonium ions are converted into ammonia gas (NH

3) by reacting with hydroxyl ions (OH-)

of excess sodium hydroxide:

((NH44))

22SOSO

44 + 2 NaOH + 2 NaOH "" 2NH

33 (gas) + Na (gas) + Na

22SOSO

44 + 2H

22OO

The ammonia distilled is dragged by bubbling steam, condensed and collected in a receiver containing an acid solution. This acid can be boric acid solution, sulfuric acid or hydrochloric acid that captures the ammonia forming solvated ammonium ions.

3. Titration: The concentration of the captured ammonium ions in the boric acid is determined by means of an acid base titration commonly using standard solutions of sodium hydroxide.

Organic nitrogen is converted into NH

4

+

NH3 is distilled and retained in a

receiver vesselNitrogen is determined

Digestion Distillation Titration

Sample

10 ReAction No.3 Reagents for food analysis

Reagents used in Kjeldahl analysis


Digestion

Kjeldahl Catalyst (Cu-Se) (1.5% CuSO4.5H2O + 2% Se) tablets, according to Wieninger

172926 1000 g (1000 tablets of 1.0 g)

3.5 kg (1000 tablets of 3.5 g)

5 kg (1000 tablets of 5.0 g)

Kjeldahl Catalyst (Cu) (6.25% in CuSO4.5H2O) tablets, accordingto Directive 93/28/EEC

174428 4 kg (1000 tablets of 4.0 g)

Sulfuric Acid 98% 173163 1000 ml, 2.5 L

Silicone antifoaming liquid 211628 100 ml, 250 ml, 500 ml

Distillation and retention of NH3

Sodium Hydroxide solution 40% w/w 171220 1000 ml, 5 L, 10 L, 25 L

Sodium Hydroxide solution 32% w/v 122666 1000 ml, 2.5 L, 5 L, 10 L, 25 L

Boric Acid solution 4% 282222 1000 ml, 5 L, 25 L

Ammonia Fixative solution 1% 283334 5 L, 25 L

Sulfuric Acid 0.25 mol/l (0.5N) 181060 1000 ml, 2.5 L, 10 L

Titration

Sodium Hydroxide 0.1 mol/l (0.1N) 181693 1000 ml, 2.5 L, 5 L, 10 L

Sulfuric Acid 0.05 mol/l (0.1N) 181061 1000 ml, 5 L, 10 L

Indicator 4.8, Mixed (Methyl Red-Bromocresol Green) 283303 250 ml

Indicator 4.4, Mixed (Methyl Red-Methylene Blue) 282430 250 ml

Methyl Red solution 0.1% 281618 100 ml

Other reagents available!

2. Sodium hydroxide solution is added to neutralize the pH and to convert NH

4

+ into NH3

1. Sample already digested with

sulfuric acid

4. NH3 is condensed

5. NH3 retained by an

acid solution (boric acid, sulfuric acid or

hydrochloric acid)

6. Finally NH3 is titrated

with sulfuric acid or sodium hydroxide

volumetric solution

3. NH3 is dragged by

bubbling steam

Kjeldahl equipment

Microbiology ReAction No.3 11

Procedure According to the ISO 6579:2002 Standard


Non-selective pre-enrichment

Buffered Peptone Water (ISO 6579:2002) (Prepared Bottles) 493795.0922 10x100 ml

Buffered Peptone Water (ISO 6579:2002) (Prepared Bottles) 493795.0981 3 x 3 L

Buffered Peptone Water (ISO 6579:2002) (Dehydrated Culture Media) 413795.1210 500 g

Selective enrichment

Rappaport-Vassiliadis (RVS) Broth (ISO 6579:2002) (Dehydrated Culture Media) 414959.1210 500 g

Tetrathionate Broth Base acc. to Muller-Kauffmann (Dehydrated Culture Media) 414961.1210 500 g

Differential isolation on Selective Agar

XLD Agar (ISO 6579:2002) (Dehydrated Culture Media) 416270.1210 500 g

XLD Agar (ISO 6579:2002) (Prepared Plate (Ø 90 mm)) 456270.0922 20 plates

Other Non-ISO Media

Chromogenic Salmonella Agar (Dehydrated Culture Media) 416110.12134 575 g

Salmonella Chromogenic Media (Prepared Plate (Ø 90 mm)) 456110.0952 10 plates

According to the ISO 6579:2002 standard, the procedure for Salmonella analysis consists in a non-selective pre-Salmonella analysis consists in a non-selective pre-Salmonellaenrichment in Buffered Peptone Water and then reseeding in two selective enrichment broths (Tetrationate according to Mueller-Kauffman and Rappaport-Vassiliadis). The next step consists in inoculating by streaking onto selective agars such as the XLD medium and others (Hektoen, Salmonella and Shigella, Chromogenic for Salmonella, etc.). The suspected colonies must be confi rmed with biochemical and serological tests.

Non-selective pre-enrichment Buffered Peptone Water ISO11Differential isolation on Selective Agar 33

4

Selective enrichment MKTTn Broth or Rappaport-Vassiliadis Broth ISO22

Chromogenic Salmonella AgarHektoen Enteric Agaretc …

Serological and Biochemical confirmation

XLD Agar ISO

S. enteritidis ATCC 13076 ATCC 13076 AIncubation at 35 ± 2ºC / 24 hours

S. enteritidisS. enteritidisS. enteritidiATCC13076

Incubation at 35ºC ± 2ºC24 hours

E.coliE.coliE.col ATCC25922Incubation at 35ºC ± 2ºC

24 hours

Determination of Salmonella sp. according to ISO 6579:2002 in food productsXLD and Salmonella Chromogenic agar

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