Why should we NOT adjust the pH of running buffer? - Would adjusting the pH cause problems during transfer/downstream steps (Jul/14/2010 )
Hello everyone,
You all are very helpful, so I am hoping I can get some great advice!
Almost every single protocol I have read to prepare running buffer for SDS-PAGE specifically states to NOT adjust the pH of the running buffer, but that it should be at about 8.0. I am wondering why we should not adjust the pH. I read somewhere that it is related to the way the gel runs, but can someone be more specific?
I am concerned because I checked the pH of my running buffer and it was around 11.0, so I adjusted it using HCl to between 8.5-9.0 and am now worried that this has affected the downstream steps. It has not happened every time, but sometimes, after colloidal gold staining of my membranes, I see a weird halo pattern (the middle region of the membrane lighter than the outside) which I had assumed was due to the orbital shaker I am using to incubate the membranes (vortex effect??), but could this be due to the lower pH of the running buffer? And sometimes the overall staining of the membrane is quite dark, making it difficult to see any bands clearly.
This is my recipe:
Tris-HEPES-SDS Running Buffer (1 L)
Tris 12.1 g
HEPES 23.8 g
SDS 1.0 g
DI water 1 L
Check pH; should be 8.0
I just feel a little lost right now since the protocols say not to adjust the pH but that it should be 8.0.... what if it isn't?? Is it worse to adjust it or just use it at 11.0, much higher than what it should be?
Any help is truly appreciated!!
if you carefully mix your buffer ingredientes in the right amounts you will get the working pH; I also learned not to check pH because titration is to avoid as the ionic strength will increase and running performance would decrease
Inmost sun on Jul 15 2010, 07:09 AM said:
Hi Inmost sun,
Thank you for the response. What you mean by "running performance" is generally how fast/slow the gel runs? I realized that quickly since the first time I remade this buffer and did NOT change the pH (kept it at 11.0), it ran MUCH faster than it should have (less than 1 hour!). This is why I decided to lower the pH in the first place.
But I want to make sure that lowering the pH only affects the speed of electrophoresis and that this could not somehow affect how the proteins transfer to the membrane. I notice afterward that the protein marker lanes look great, very straight and clear, and even after transfer, they look fine. It's just after staining with colloidal gold that I get this halo pattern on the membrane, but only sometimes...
Do you not adjust the pH because there is SDS in the buffer? I remember during my PhD that an RA told me never to pH SDS (but I can't remember at what concentration) - because it screws up the readings?
??
Just a thought...
Clare
alerz on Jul 15 2010, 12:10 AM said:
You all are very helpful, so I am hoping I can get some great advice!
Almost every single protocol I have read to prepare running buffer for SDS-PAGE specifically states to NOT adjust the pH of the running buffer, but that it should be at about 8.0. I am wondering why we should not adjust the pH. I read somewhere that it is related to the way the gel runs, but can someone be more specific?
I am concerned because I checked the pH of my running buffer and it was around 11.0, so I adjusted it using HCl to between 8.5-9.0 and am now worried that this has affected the downstream steps. It has not happened every time, but sometimes, after colloidal gold staining of my membranes, I see a weird halo pattern (the middle region of the membrane lighter than the outside) which I had assumed was due to the orbital shaker I am using to incubate the membranes (vortex effect??), but could this be due to the lower pH of the running buffer? And sometimes the overall staining of the membrane is quite dark, making it difficult to see any bands clearly.
This is my recipe:
Tris-HEPES-SDS Running Buffer (1 L)
Tris 12.1 g
HEPES 23.8 g
SDS 1.0 g
DI water 1 L
Check pH; should be 8.0
I just feel a little lost right now since the protocols say not to adjust the pH but that it should be 8.0.... what if it isn't?? Is it worse to adjust it or just use it at 11.0, much higher than what it should be?
Any help is truly appreciated!!
clare is correct. sds will clog the porous plug of the pH electrode when it precipitates as kds. this will foul the reading.
as inmost sun said, if you prepare the buffer carefully, weighing the correct amount and using the correct form (hydration) of the components then you don't have to worry about the pH.
if you insist on checking the pH then do so before adding the sds, but don't adjust it.
Thanks for the explanation - nice to know why
Clare
mdfenko on Jul 16 2010, 08:59 PM said:
as inmost sun said, if you prepare the buffer carefully, weighing the correct amount and using the correct form (hydration) of the components then you don't have to worry about the pH.
if you insist on checking the pH then do so before adding the sds, but don't adjust it.