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Proclin preservative

Proclin preservative - ELISA Assay Forum

Proclin preservative - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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  #1  
Old 01-14-2011, 12:04 AM
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Default Proclin preservative



Has anyone use Proclin as a preservative in their ELISA buffers and if so does it work ok?
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  #2  
Old 01-26-2011, 01:17 PM
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Default Re: Proclin preservative

From the way of the question it would seem that you have ProClin used but with poor results. ProClin series are broad spectrum preservatives that when in bacteria & fungi contact show bacteriostatic activity and shortly after microbicides effect. The action mechanism of two components of the product, 5-chloro-2-methyl-4-isothiazolin-3-one (76.67 %) and 2-methyl-4-isothiazolin-3-one (23.33 %), to reside both on there ability to quickly penetrate cell membranes and to inhibit specific enzymes in the Krebs cycle (pyruvate dehydrogenase, alfa-ketoglutarate dehydrogenase, succinate dehydrogenase, and NADH dehydrogenase), having as resulted decrease of ATP synthesis. At low concentrations (< 40 ppm) ProClin show quickly bacteriostatic activity while higher concentrations (40 -100 ppm) exhibit rapid microbicidal effects, only if bacteria charge is inclusive among 102 and 103, if this it’s greater, the preservative concentration has to proportionally be more. ProClin, biocidal isothiazolones, exhibit a wide range of chemical compatibility and pH tolerance, but one class of chemicals that is aggressive to the isothiazolone molecules is secondary amines. With their strong nucleophilic activity, secondary amines break the isothiazolones’ sulfur-nitrogen bond, resulting in ring-opening and loss of biocidal activity. Much of this interaction is pH-dependent; at alkaline pH the equilibrium favors amines that are highly aggressive nucleophiles. At neutral pH and below, the amines are protonated and have reduced nucleophilic activity. In clinical chemistry or immunometric assays (EIA) the often used buffers belong at primary amines (methylamine, ethanolamine [2-aminoethanol], Tris and TES) or secondary amines (dimethylamine, methylethanolamine and HEPES [containing both positive and negative ionizable groups; chemical characteristics that do it belong at intermediary group between secondary and tertiary amines]), which from pH 7.2 the biocides ability is progressively reduced, especially if store at room temperature. For to maximize biocides ProClin activity we suggest : a) adjust pH solution at 7.0, filter solution on 0.45 m filter, to strongly reduction bacterial charge, and then add 2.86 ml/dl (0.1%) of ProClin 300; b) do not use buffers containing primary or secondary amines, especially if you job at critical pH, but buffers based on sterically hindered as tertiary amines that are devoid of hydroxy groups or other weak donors on the α, β, or γ carbons, and are incapable of forming even weak complexes with metal ions. To belong to this category the alkyls and the alkylsulfonates derived of the morpholine (e.g. MOPS - B tertiary amines group), piperazine (e.g. PIPES - A tertiary amines group), ethylenediamine and methylenediamine. With the exception of the morpholine derivatives, these compounds have two protonatable sites and, therefore, can be used to buffer two separate pH regions with range inclusive from 3 to 11; c) When primary or secondary amine buffers are optimal for the method, even at critical pH condition, then add to the buffers a Proclin 300 concentration ≥ at 40 ppm (1.33 ml/L peer to 0.00465%) after to have added 0.025-0.1 mg/ml neomycin sulfate and 0.9-1 mg/ml NaN3.
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Old 01-27-2011, 06:14 AM
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Default Re: Proclin preservative

Sorry I should have explained a bit better, I work in a diagnostic ELISA lab and we make up our own buffers. Currently we want to stop using sodium azide as a preservative and want to try a "friendlier" one and I found Proclin on the web and was wondering if anybody else was using it in their in house made buffers and did they like it. I have found some commercial pre-made ELISA buffers have it in already. ELISA buffers are generally at a pH of 7.4 with the exception of the coating and substrate buffers (pH 9.6 and 9.8 respectively). Thanks for the information.
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Old 01-27-2011, 09:00 AM
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Default Re: Proclin preservative

Tell me what buffers you use and I will tell you what ProClin type to use and specific concentration
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Old 01-27-2011, 11:31 PM
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Default Re: Proclin preservative

These are the regular buffers I make up every week;

Carbonate coating buffer (1L:1.59g Na2CO3, 2.93g NaHCO3, pH 9.6)
Phosphate buffered saline (PBS) 10X stock (1L: 80g NaCl, 2g KH2PO4, 11.4g Na2HPO4, 2g KCl, pH 6.7)
Wash Buffer (1L: 100ml PBS stock, 0.5ml Tween 20, 1g skim milk powder)
Extraction buffer (1L: 100ml PBS stock, 20g PVP, 2g BSA, 1.3g Na2SO3, 20g Tween 20, pH 7.4)
Conjugate buffer (1L: 100ml PBS stock, 20g PVP, 2g BSA, pH 7.4)
Substrate buffer (1L: 97ml diethanolamine, 0.05g MgCl2, pH 9.8)
The last one probably needing the higher concentration I guess.
Thanks for all your help!
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Old 02-01-2011, 03:56 PM
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Default Re: Proclin preservative

Dear Aldann
following is described what from you asked :
CARBONATE COATING BUFFER
This solution is a strong isothiazolone molecules deactivating therefore, ProClin employment as preservative it is useless, while azides (0.2 g/L) are strongly active. Nevertheless, the coating could happen without preservative, provided that blocking solution containing ProClin, for saturate free sites, is used. To realize this is necessary know nature molecule coating.
POSHATE BUFFER SALINE (PBS)
Stock Solution 10x : 80 g/L NaCl, 2 g/L KH2PO4, 11.4 g/L Na2HPO4 anhydrous, 2 g KCl at pH 6.7, it has to contain a ProClin 300 (concentration 3.49 %) concentration between 0.05 and 0.08 % per solution 1x.
Realization procedure
In graduate beaker to put 600 ml of ddH2O, dissolve 80 g/L NaCl, 2 g/L KH2PO4, 11.4 g/L Na2HPO4 anhydrous, 2 g KCl, and adjust at pH 6.7 with acid or alkaline phosphate and mix for 30 mins. Add 18.018 ml (0.063 %) ProClin 300, ad fill to 1 liter with ddH2O and mix for 20 min. Aliquote in clear plastic bottle and store at 4 C. Stable per several months.
WASHER BUFFER
In graduate beaker to put 300 ml of ddH2O, add 100 ml PBS stock solution, 0.5 ml Tween 20, dissolve 1 g skim milk powder and adjust pH at 7.2 0.2, if needed, add 1.06 ml Proclin 300, for to get an 0.1 % final concentration, fill to 1 liter with ddH2O and mix for 30 mins. Aliquote in clear plastic bottle and store at 4 C. Stable per several months.
EXTRACTION BUFFER
In graduate beaker to put 600 ml ddH2O, add 100 ml PBS stock solution, dissolve 20 g PVP, 2 g BSA, 1.3 g Na2SO3, 20 g Tween-20, adjust pH at 7.4, if needed, and add 1.06 ml Proclin 300, for to get an 0.1 % final concentration, fill to 1 liter with ddH2O and mix for 30 mins. Aliquote in clear plastic bottle and store at 4 C. Stable per several months.
CONIUGATE BUFFER
Dilute in 600 ml ddH2O, 100 ml PBS stock solution, dissolve 20 g PVP, 2 g BSA, what it has the useful final ProClin 300 concentration 0.063 %, fill to 1 liter with ddH2O and mix for 30 mins. Aliquote in clear plastic bottle and store at 4 C. Stable per several months.
SUBSTRATE BUFFER
Component
a) Buffer Diethanolamine (DEA) : is a secondary amine it would go replaced with tertiary amines such as 99 % triethanolamine (TEA), however if this isn't possible then it needs minimize levels of DEA in your substrate buffer between 15 – 60 mM, preferably about 25-30 mM;
b) Cofactor agent : use MgCl2 at high concentration 1-10 mM, preferably about 4.5-6.5 mM;
c) Stabilizing agent : use ZnCl2 at low concentration 0.05-2.0 mM, preferably about 0.08-0.13 mM;
d) Solubilizing agent : having synergic effect against gram negative bacteria, such as quaternary ammonium detergent or non ionic surfactant such as Tween-20 that is better at low concentration between 0.01-0.03 % (v/v) preferably about 0.015-0.025 %;
e) Preservative agent : ProClin 300 it has to be among 0.02-0.08 %.
Realization procedure
In graduate beaker to put 300 ml of ddH2O, and pipette in continue mix, 2.9 ml (30 mM) of DEA, then adjust at pH 9.8 with 1 M HCl at 25 C. Dissolve 0.514 g (5.4 mM) MgCl2 anhydrous and 17 mg (0.124 mM) ZnCl2, and mix for 15 mins. Check again pH, and add 0.22 ml Tween-20, continue to mix, without foam, not less than 30 mins and add 15.2 ml (0.053 %) Proclin 300. Fill to 1 liter with ddH2O, mix for 30 mins and after aliquote in clear plastic bottle and store at 4 C. Stable for several months.
If you change TEA instead DEA, I can give to you protocol for to realize pNPP substrate stable for 18 months at 4 C in liquid form.
“always available for the one whom asks”
Good luck and let me know
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  #7  
Old 02-07-2011, 01:24 AM
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Default Re: Proclin preservative

Thanks so much for your time and help!
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