I don't believe at all that 50% glycerol prevents bacterial and fungal growth in long term (e.g. overnight or over weekend) incubations with antibodies, and how to handle those slimy solutions. I have used Thimerosal (=Tiomersal) with great success as an alternative for along time, but whatever contains mercury (and tiomersal contains quite a lot, even that it is a WHO-recommended additive to baby vaccines!) is now forbidden to use in Sweden. So, I would be more than happy to learn about proper alternatives to sodium azide. The latter is perhaps not so terribly bad at low concentrations since some enterprises (e.g. Dako) even sell their HRP-antibody conjugates in solutions containing 15mM sodium azide. Unfortunately, I do not know precisely about the inhibition process of azide at HRP. However, since HRP is related to cytochrome-C it contains a iron-heme-group; azide is known to inhibit cytochrome oxidase by binding irreversibly to the heme-iron. My only proper experience is, that direct comparisons of low-conc. azide-poisoned HRP-enzyme activities with "non-poisonend" HRP activities show that the latter HRP preparations work much faster at approx. the same dilutions, telling me that azide is not a competitive inhibitor of HRP but rather a permanent/irreversible, of course, depending on the initial inhibitor concentration. Haven't worked that out in proper enzyme-kinetics tests so far, unfortunately!

So, Jalal, what precisely does "ProClin" and where to get it suitably, Sigma? 0.02% sounds pretty much the same concentration as often used for azide. As a mechanism of ation I only found on the web: The active components of ProClin are three isothiazolones, 2-Methyl-4-isothiazolin-3-one, 5-Chloro-2-methyl-4-isothiazolin-3-one and 1,2-Benzisothiazolin-3-one. Which formulation of ProClin 150, 200, 300, 950 containing which stabilisers do you recommend when working with HRP???

Thanks to all of you for any suitable answers!!

50% Glycerol can be used as preservant. U can go to Jackson Immuno Research site and find alternative dilutents for antibodies and blocking solutions.

Very useful advice. Thank you very much

We also use Thimerosal instead of sodium azide. At 0.1% should be fine

You can use ProClin preservatives at concentrations as low as 0.02%.

@Priyabrata Mohanty: are you sure that glycerol is used to prevent growth of bacteria? I thought that it is used as cryoprotectant.

I don't believe at all that 50% glycerol prevents bacterial and fungal growth in long term (e.g. overnight or over weekend) incubations with antibodies, and how to handle those slimy solutions. I have used Thimerosal (=Tiomersal) with great success as an alternative for along time, but whatever contains mercury (and tiomersal contains quite a lot, even that it is a WHO-recommended additive to baby vaccines!) is now forbidden to use in Sweden. So, I would be more than happy to learn about proper alternatives to sodium azide. The latter is perhaps not so terribly bad at low concentrations since some enterprises (e.g. Dako) even sell their HRP-antibody conjugates in solutions containing 15mM sodium azide. Unfortunately, I do not know precisely about the inhibition process of azide at HRP. However, since HRP is related to cytochrome-C it contains a iron-heme-group; azide is known to inhibit cytochrome oxidase by binding irreversibly to the heme-iron. My only proper experience is, that direct comparisons of low-conc. azide-poisoned HRP-enzyme activities with "non-poisonend" HRP activities show that the latter HRP preparations work much faster at approx. the same dilutions, telling me that azide is not a competitive inhibitor of HRP but rather a permanent/irreversible, of course, depending on the initial inhibitor concentration. Haven't worked that out in proper enzyme-kinetics tests so far, unfortunately!

So, Jalal, what precisely does "ProClin" and where to get it suitably, Sigma? 0.02% sounds pretty much the same concentration as often used for azide. As a mechanism of ation I only found on the web: The active components of ProClin are three isothiazolones, 2-Methyl-4-isothiazolin-3-one, 5-Chloro-2-methyl-4-isothiazolin-3-one and 1,2-Benzisothiazolin-3-one. Which formulation of ProClin 150, 200, 300, 950 containing which stabilisers do you recommend when working with HRP???

Thanks to all of you for any suitable answers!!

Dear Heinrich,

I was also told about thimerosal so I will try this. I was puzzled because I need to make stock solutions in bulk quantities and in the same time avoid sodium azide. Therefor, thanks a lot for such an informed input. I am still looking for ProClin

Preservatives made of mixtures (in the ratio of approximately 3:1) between 5-chloro-2-methyl-4-isothiazolin-3-one, and 2-methyl-4-isothiazolin-3-one are commercially available from Rohm & Haas under various tradenames, such as KATHON CG, PROCLIN 300 and PROCLIN 150, which vary from one another by the stabilizer used in formulating the preservative mixture and other factors not affecting the active ingredients (glycol). This family of products is a member of the class of preservatives known as isothiazolines.  ProClin preservative does not affect the functionality of most enzyme- or antibody-linked reactions, and will not interfere with assay indicators.

As for mechanisms of action, ProClin preservatives attack the central metabolic cycle of the cell, the Krebs cycle, at three specific enzyme sites: α-ketoglutarate dehydrogenase, succinate dehydrogenase, and NADH dehydrogenase. Other inhibited enzymes include pyruvate dehydrogenase, which is vital for the oxidation

of pyruvate to acetyl-CoA; as well as lactate dehydrogenase and alcohol dehydrogenase, which detoxify waste by-products synthesized during

fermentation. With cellular metabolism debilitated, microorganisms rapidly lose their ability to produce ATP and carry on vital functions, such as transcription, translation and damage repair. All bacteria and fungi possess at least some parts of the Krebs cycle, thus ProClin preservatives represent a highly efficient broad spectrum biocide.

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/1/proclin-300-preservative-for-diagnostic-reagents.pdf

Hi,

Is there any restrictions on the upper limit of Proclin that can be used. I found a post in an online forum stating that 0.1% would require products to be labelled as hazardous but I can't find any supporting information.

Thanks,

Alan

I also want an alternative to sodium azide to preserve haemolysed haemoglobin samples used for cellulose acetate electrophoresis HB genotype controls.