Molecular Biology

buffer components

 

 

 

Introduction to Buffers

Most applications in biotechnologies and biochemistry of proteins operate in aquous solutions.

Water is determinent to interactions of biological systems, dissociating in H+ and OH- ions thus interfering with ions and charged biomolecules, but also interacting by Van de Walhs binding to solvate biomolecules and by hydrophobic interactions to form micelles or precipitates...

To that point, buffers are aquous solutions containing partly neutralized weak acids or bases that show little change in pH (H+ concentration) whatever ions are added. Requirements should be considered for the choice of the buffer, and it’s use in each application. I.e. the pH should be determined at the final temperature, in presence of salts (i.e. phosphate pH change with salts concentration) near the pKa of the buffering compound.

The buffering compound should not absorb at wavelenghts (i.e. at 240-270nm for mass spectrometry)… Beside mineral buffers that are widely used, althought their pKa do not always suit the best, one might consider biological buffer that have several advantages, and are also proposed with Uptima UltraPure quality.

Biological buffers differ from classic mineral to several points: they have pKa values at or near physiological pH (between 6 and 8). These buffers are non toxic to cells, and are not absorbed through cell membranes. The concentration, temperature, and ionic composition of the medium has minimal effect on the buffering capacity. They are resistant to enzymatic and non enzymatic degradation, furthermore they are essentially transparent to visible and ultraviolet light.

 

Ammonium Persuflate

UP306098

Boric Acid

UP070440

1 kg

ACS Grade

H3BO3

CAS [10043-35-3]

MW 61.83

pKa1(25°C)=9.24

DNase, RNase, Proteases free

Purity 99.5 %

Borate buffered saline should not be used in the presence of polyols, including carbohydrates and their derivatives with which they may chelate compounds. Borate buffers also have a high bacteriocidal effect. The use of borate buffers in gel electrophoresis of proteins can result in spreading of the zones.

Citric acid

UP168781

1kg

C6H8NO7

CAS [77-92-9]

MW 192.13

pKa1(25°C)=3.01 , pKa2(25°C)=4.76

pKa3(25°C)=5.40

Purity: >99%

DNase RNase, Proteases free

Citrate is used notably for elution in affintiy chromatography, but also for cell media.

Detergents

DTT (1,4-Dithiothreitol)

UP284250

1g

UP284255

5g

C4H10O2S2

CAS [27565-41-9]

MW 154.25

Free sulfhydryl group content >99.4%

Disulfide content <0.5%

DNase, RNase, Protease activity : None detected

Technical Sheet - MSDS

Alternative product : TCEP - UP24221

DTT is an important chaotropic agent used to destabilize disulfide bonds and disrupt tertiary structure in proteins.

While it is an effective protein denaturant at high concentrations, at low concentrations however, it acts to maintain native sulfhydryl groups, thereby preventing conformational changes at important active sites. The reducing power of DTT therefore makes it useful for protein structural analysis as well as for preserving biological activity in enzymes, antibodies, and growth factors.

Uptima’s Biotechnology Grade DTT has particularly high purity (free sulfhydryl content > 99.4%) and very low UV absorptivity, important for spectrophotometric analysis. These qualities, coupled with a high solubility in water, make Uptima DTT an outstanding choice for your molecular biology applications.

EDTA, Disodium Salt

UP036290

1kg

(EthyleneDiamineTetraAcetic acid) - Biotechnology grade

C10H14N2O8Na2. 2H20

CAS [6381-92-6]

MW 372.24

Suggested Starting Concentration : 0.2 - 0.5 mg/ml (0.5-1.3mM)

DNase, RNase : None detected

Chelator of divalent cations.

Inhibits enzymes, such as metalloproteases, that require divalent cations for activity.

PBS Buffer

SDS, powder

UP649100

500g

SDS, 20% solution

UP896826

500ml

-

UP896827

2x500ml

Sodium Dodecyl Sulfate ; biotechnology grade

Purity > 99.0% ; C12 Content > 99.0%

DNase, RNase, Protease free

OD 260 and OD 280 (3% solution in water) > 0.1

Sodium Dodecyl Sulfate is a critical reagent in many molecular biology applications. It is widely known that the purity and C12 content dramatically affect the performance of this detergent. For example, contaminating levels of C16-alkyl sulfate particularly affect protein renaturation, and contaminating UV absorbing materials affect detection sensitivity. Additionally, heavy metals/chloride contaminants affect separation and enzymatic activities.

Biotechnology Grade SDS is especially high in both purity and C12 content. This nuclease and protease free material is ideally suited for nucleic acid purification, hybridization cocktails, electrophoresis, wash buffers and protein studies.
Sucrose

UP252031

1kg
Ultra Pure Grade

C12H22O11

CAS [57-50-1]

MW 342.3

Purity 99.9 %

DNase, RNase None detected

TBS Buffer

Tweenâ20

TAE Buffer

TBE Buffer

TG-SDS Buffer

Formamide

UP07099

Glycine

UP018225

1kg

Biotechnology Grade

C2H5NO2

CAS [56-40-6]

MW 75.07

Purity > 99.0 %

A280nm <0.15, A26nm <0.03

Glycine is used notably for elution in affintiy chromatography, in relectrophoresis buffers and in biochemistry (i.e. as quenching agent).

 

 

HEPES

UP061940

250g

2-[4-(2hydroxyethyl)-1piperazinyl]-ethanesulfonic acid

C8H18N2O4S

CAS [7365-45-9]

MW 238.3

pKa(25°C)=7.5

Purity >99%

A280(1M, water) < 0.1

DNase RNase, Proteases free

Useful pH range is 6.8-8.2

 

MOPS

UP062000

100g
Ultra ACS Grade

3-(N-Morpholino Propane Sulfonic acid)

C7H15NO4S

CAS [1132-61-2]

MW 209.27

pKa(25°C)=7.2

MOPS is used for RNA electrophoresis in agarose. It is available in a free acid form, and works exceptionally well for formaldehyde gels at a 20mM concentration. The free acid form must be adjusted to the working pH with an appropriate base such as sodium hydroxide, potassium hydroxide, or tetramethylammonium hydroxide.

PIPES

UP061980

100g

UP091981

250g

Piperazine-N,N¢-bis(2-ethane-sulfonic acid); 1,4-Piperazinediethanesulfonic acid

C8H17N2O6S2.1.5Na

CAS [10010-67-0]

MW 335.37

pKa(25°C)=6.8

Purity (Anhydrous) 99.0 %

DNase, RNase, proteases free

Buffering range is 6.1 - 7.5 (at 25°C)

PIPES is a member of the ethanesulfonic acid buffer series, first introduced by Good et al., developed to meet certain criteria: midrange pKa, maximum water solubility and minimum solubility in all other solvents, minimal salt effects, minimal change in pKa with temperature, chemically and enzymatically stable, minimal absorption in visible or UV spectral range and easily synthesized.1 Since its pKa at 37°C is near physiological pH, it has applications in cell culture work. Buffers can be prepared by adding a solution of base to PIPES free acid, titrating to the appropriate pH, or by mixing equimolar solutions of the monosodium salt and the disodium salt, titrating to the appropriate pH.

Applications:

Glutaraldehyde fixation of plant and animal tissue samples can cause loss of lipid, leading to apparent morphological changes. Lipid loss and artifacts are minimized when PIPES was used to buffer the glutaraldehyde fixative.

Alkaline phosphatase activity is lost selectively from certain rat hepatocyte organelles when fixed for ultracytochemistry with cacodylate-buffered glutaraldehyde. When PIPES was used as buffer, retention of activity was 60% greater. Fixation of fungal zoospores for fluorescence microscopy and electron microscopy was optimal with a combination of glutaraldehyde and formaldehyde in PIPES buffer.

Temed

Tris HCI

UP09154D

500g

-

UP09154E

1kg

-

UP09154F

5x1kg

Tris(hydroxymethyl) aminomethane hydrochloride

C4H11NO3.HCl

CAS [1185-53-1 ]

MW 157.64

pH (0.1M, Water) @25°C 4.2-4.9

Urea

UP031903

500g

Ultra Pure Grade

UP031904

1kg

CH4NO2

CAS [57-13-6]

MW 60.06

Used for the denaturation of proteins and as a mild solubilization agent for insoluble or denatured proteins. May be used with guanidine hydrochoride and dithiothreitrol (DTT) in the refolding of denatured proteins into their native or active form.

Purity > 99.5%

DNase, RNase, protease free

 

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