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sudheer sangeetham | 3 Jan 2012 17:52
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10X PBS

Hi People

I have few doubts regarding phosphte buffer saline (PBS). Recently I found
one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
not KCl, am I right?

I have prepared the buffer like this

NaH2PO4 ( 0.038M)
Na2HPO4.2H20 ( 0.162M)
NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
did I do any mistake....

Could any one please tell me did i do any mistake?

Thank you in advance

--

-- 
Sudheer Babu.S
Ph.D student
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.
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Hiranya Roychowdhury | 3 Jan 2012 21:08

RE: 10X PBS

Since you say you have few questions, I am assuming that you are quite certain about your composition.  PBS
stands for "Phosphate buffered saline" so it is NaCl, not KCl.  Usually we use Na-salts for the phosphate
combination.  Potassium phosphate and -biphosphate may be used, but for all practical purposes it is Na-
that is preferred since lot of compounds do not go well with K+

Hiranya S. Roychowdhury, Ph.D.
Associate Professor
Health & Public Services
NMSU-Dona Ana Community  College
575 527 7725 (office)

________________________________________
From: methods-bounces@...
[methods-bounces@...] on behalf of sudheer
sangeetham [sudheer.pbm07@...]
Sent: Tuesday, January 03, 2012 9:52 AM
To: Methods@...
Subject: 10X PBS

Hi People

I have few doubts regarding phosphte buffer saline (PBS). Recently I found
one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
not KCl, am I right?

I have prepared the buffer like this

NaH2PO4 ( 0.038M)
Na2HPO4.2H20 ( 0.162M)
(Continue reading)

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Irit Rappley | 3 Jan 2012 22:09
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Re: 10X PBS

The pH of the 10X solution can be slightly different from the pH of the 1X solution (but not usually  as big as
what you are seeing, I think). So you should check the pH of your 1X solution and adjust accordingly. 

When I make PBS 10X stock, the pH is often wrong and I simply adjust it with 5M NaOH. Maybe your calculation did
not take into account the pH of the water (usually ~5 or 6, definitely not 7)?

Hope that helps,
Irit

On Jan 3, 2012, at 12:08 PM, Hiranya Roychowdhury wrote:

> Since you say you have few questions, I am assuming that you are quite certain about your composition.  PBS
stands for "Phosphate buffered saline" so it is NaCl, not KCl.  Usually we use Na-salts for the phosphate
combination.  Potassium phosphate and -biphosphate may be used, but for all practical purposes it is Na-
that is preferred since lot of compounds do not go well with K+
> 
> 
> Hiranya S. Roychowdhury, Ph.D.
> Associate Professor
> Health & Public Services
> NMSU-Dona Ana Community  College
> 575 527 7725 (office)
> 
> ________________________________________
> From: methods-bounces@...
[methods-bounces@...] on behalf of sudheer
sangeetham [sudheer.pbm07@...]
> Sent: Tuesday, January 03, 2012 9:52 AM
> To: Methods@...
> Subject: 10X PBS
(Continue reading)

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DK | 4 Jan 2012 00:07

Re: 10X PBS

In article <mailman.69.1325615292.3721.methods@...>,
sudheer sangeetham <sudheer.pbm07@...> wrote:
>Hi People
>
>I have few doubts regarding phosphte buffer saline (PBS). Recently I found
>one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
>Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
>not KCl, am I right?
>
>I have prepared the buffer like this
>
>NaH2PO4 ( 0.038M)
>Na2HPO4.2H20 ( 0.162M)
>NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
>did I do any mistake....

1. Measurng pH in ~ 2 M NaCl is not going to be very meaningful with 
most electrodes. 
2. pKa is a function of ionic strenght - no surprise that 10X and 1X have 
different pH.
3. Dilute 10X, measure pH and if it's not above 7.2, you either made 
an error making the solution or pH electrode is not measuring correctly. 
4. The ratio you used above is for 0.2 M sodium phosphate, another 
10X away from your 20 mM. (The difference between 200 and 20 will
be small but not negligible). 

5. "PBS" can stand for many things. Cell culture PBS solutions
contain not ony some KCl (around 10 mM) but also some divalents,
CaCl2 and/or MgCl2.
(Continue reading)

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Jayakumar, R | 3 Jan 2012 22:21
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RE: 10X PBS

You might be referring to the use of PBS in animal systems like mice or rat.  When they mean PBS i.p. (was it
written this way?), it means administration of PBS into animal was via intraperitoneal injection or i.p
(in the abdmomen).   Unless otherwise told, PBS contains both KCl and NaCl along with Na2HPO4 and NaH2PO4. 
Refer the Sambrook manual or the current protocols in Molecular biology or any standard tissue culture
manual for the  PBS recipe.  When the monobasic and dibasic salts are corrected added, the pH will be around
7.5.  If it is 6.4, your recipe is not right.
Jay

-----Original Message-----
From: methods-bounces@...
[mailto:methods-bounces@...] On Behalf Of
sudheer sangeetham
Sent: Tuesday, January 03, 2012 11:53 AM
To: Methods@...
Subject: 10X PBS

Hi People

I have few doubts regarding phosphte buffer saline (PBS). Recently I found
one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
not KCl, am I right?

I have prepared the buffer like this

NaH2PO4 ( 0.038M)
Na2HPO4.2H20 ( 0.162M)
NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
did I do any mistake....
(Continue reading)

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WS | 4 Jan 2012 09:59
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Re: 10X PBS

Hi, I'm using this recipe for PBS, suitable for many occasions.

Actually, it is a rinsing buffer for cells, prior to trypsinization (D-
PBS from PAA), hence no divalent cations like Calcium and Magnesium.

Depending on your application (when you don't apply it on living
beings e.g.) , you may replace the potassium salts with sodium salts
for simplicity

The mean thing is that one needs to be careful about the actual
molecular weights of the phosphates as they are available with varying
crystal water content, so you might double check the formula and the
molecular weight on the actual box.

1xPBS:

KCl		M= 74,6 g/mol	0.2g/l 	→ 2,68 mM
NaCl		M= 58.4 g/mol	8g/l	→ 137 mM
KH2PO4	M= 136.1g/mol	0.2g/l	→ 1.47 mM
Na2HPO4	M= 142g/mol		1.16g/l	→ 8.17 mM

Formulation, 10x Concentrate (g/l)
Inorganic Salts

KCl
2.0
KH2PO4
2.0
NaCl
80.0
(Continue reading)

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David-Paul Minde | 4 Jan 2012 13:11
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Re: 10X PBS - better add KCl

indeed adding KCl to your phosphate buffer is highly recommended - and
essential for keeping (most precious) proteins "alive" during
freeze-thaw cycles (with NaCl only, phosphate buffer will precipitate
and solution turns acidic - pH about 3 or so ...)
cheers,
David Minde
2012/1/3, Jayakumar, R <R.Jayakumar@...>:
> You might be referring to the use of PBS in animal systems like mice or rat.
>  When they mean PBS i.p. (was it written this way?), it means administration
> of PBS into animal was via intraperitoneal injection or i.p (in the
> abdmomen).   Unless otherwise told, PBS contains both KCl and NaCl along
> with Na2HPO4 and NaH2PO4.  Refer the Sambrook manual or the current
> protocols in Molecular biology or any standard tissue culture manual for the
>  PBS recipe.  When the monobasic and dibasic salts are corrected added, the
> pH will be around 7.5.  If it is 6.4, your recipe is not right.
> Jay
>
>
> -----Original Message-----
> From: methods-bounces@...
> [mailto:methods-bounces@...] On Behalf Of
sudheer sangeetham
> Sent: Tuesday, January 03, 2012 11:53 AM
> To: Methods@...
> Subject: 10X PBS
>
> Hi People
>
> I have few doubts regarding phosphte buffer saline (PBS). Recently I found
> one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
(Continue reading)

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Jayakumar, R | 4 Jan 2012 15:24
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RE: 10X PBS

Ideally the pH should not change much between 10X and 1X if deionised water was used for dilution.  Presnce of
NaCl has nothing to do with pH changes since it contributes neither hydroxyl nor hydrogen ions (unless
otherwise it is contaminated with something else), the balance of which determines pH (not just ionic
strength).  The balance between the monobasic and dibasic is thus maintained the same whatever dilution
the stock is diluted to.  If stock concentrations affected pH, many companies that make 10X buffer stocks
will be out of business.  During dilutions make sure to use deionised water (Millipore MilliQ for example)
or the ions in water can slightly affect the pH, but not that significantly as you observed. 
Jay

-----Original Message-----
From: methods-bounces@...
[mailto:methods-bounces@...] On Behalf Of DK
Sent: Tuesday, January 03, 2012 6:08 PM
To: methods@...
Subject: Re: 10X PBS

In article <mailman.69.1325615292.3721.methods@...>,
sudheer sangeetham <sudheer.pbm07@...> wrote:
>Hi People
>
>I have few doubts regarding phosphte buffer saline (PBS). Recently I found
>one article related to my work, in that they used PBS ( Nacl/iP ) pH-7.4.
>Nacl/iP does it mean, it should contain Na2HP04 and NaH2PO4 and Nacl but
>not KCl, am I right?
>
>I have prepared the buffer like this
>
>NaH2PO4 ( 0.038M)
>Na2HPO4.2H20 ( 0.162M)
>NaCl (1.5 M ) but when i check the pH it was showing 6.4, is it correct?
(Continue reading)

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DK | 4 Jan 2012 21:38

RE: 10X PBS

In article <mailman.75.1325703460.3721.methods@...>,
"Jayakumar, R" <R.Jayakumar@...> wrote:
>Ideally the pH should not change much between 10X and 1X if deionised water was
> used for dilution.  Presnce of NaCl has nothing to do with pH changes since it
> contributes neither hydroxyl nor hydrogen ions 

You need to refresh your physical chemistry. 
(Hint: cations compete with protons for binding to anions) 

DK
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Joshua Silverstein | 4 Jan 2012 22:07
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Re: 10X PBS

You don't need physical chemistry, just general.

If you take 10X PBS and dilute it with deionized water, it will certainly
change the pH.  For a 10-fold dilution, it should change the pH by
approximately 1 (log scale).

On Wed, Jan 4, 2012 at 3:38 PM, DK <dk@...> wrote:

> In article <mailman.75.1325703460.3721.methods@...>, "Jayakumar,
> R" <R.Jayakumar@...> wrote:
> >Ideally the pH should not change much between 10X and 1X if deionised
> water was
> > used for dilution.  Presnce of NaCl has nothing to do with pH changes
> since it
> > contributes neither hydroxyl nor hydrogen ions
>
> You need to refresh your physical chemistry.
> (Hint: cations compete with protons for binding to anions)
>
> DK
>
> _______________________________________________
> Methods mailing list
> Methods@...
> http://www.bio.net/biomail/listinfo/methods
>
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