Information

PBST vs. TBST buffer in western blotting


What is the advantages and disadvantages of using either PBST or TBST in western blotting, or while working with proteins in general? Are there other buffers which are also used for western blotting, or washing steps while working with proteins?


I used to work for a company well-known for its modification state-specific antibodies, including phospho-specific ones, and they actually performed extensive in-house testing of PBST vs. TBST in Western blotting. Part of the reason the company chose to recommend the use of Tris-buffered saline over phosphate-buffered saline based buffers was the clearly-demonstrated fact that some phospho-specific antibodies just didn't work as well in PBST as they did in TBST, for whatever reason. As I recall (it's been quite a while since the testing was performed), some of the antibodies tested gave weaker signals, with more background, and one in particular just didn't work at all in PBST - no signal whatsoever. For that reason, the company chose to standardize its recommended Western blotting protocol with TBST, and it's been that way ever since.

That being said, I'm sure that the majority of antibodies out there would work just fine in either buffer. However, to be on the safe side, if a company states one buffer or other in their protocol, it would be best to use the recommended one.


Washes for Western, PBS or TBST? - (Mar/13/2006 )

Hi I'm just curious if people do their post primary antibody pre-detection washes of a blotted PVDF membrane in PBS or TBST, and if one rather than the other, why?

if you're looking at phosphorylation state, you HAVE to use TBS (the phosphates in PBS will screw up your results)

this is the main difference of which I am aware

hi,
aimkins in our lab we use, phospho ERK, jnk n p38 antibodies, every body uses PBS.
I wondering about ur answer, if possible please make me clear in this concept.
thanks

if you're looking at phosphorylation state, you HAVE to use TBS (the phosphates in PBS will screw up your results)

this is the main difference of which I am aware

those are the same Westerns that we do. I was told that TBS would screw it up and give you high background, as well as potentially interfere with your results.

I do not have a reference however I have heard it from several people. Perhaps it's an old-wives' tale?

I use 6x TBST before adding my secondary, and 5xTBST then 1x PBS before detection. I was told that the tween can give high backgrounds and to wash it off with PBS before beginning the detection.

We wash 3X's in TBS-T and 1X PBS and it works fine!

those are the same Westerns that we do. I was told that TBS would screw it up and give you high background, as well as potentially interfere with your results.

I do not have a reference however I have heard it from several people. Perhaps it's an old-wives' tale?

I use PBS. I will look for phosphorylation state soon, so i'm interested in what you said. I also would like to ask if somebody has used anti phosphoserine antibodies to detect phosphorilated proteins. If so any recommendations? Is it also possible to quantify and compare phosphorylation state using this antibodies
Thanks

those are the same Westerns that we do. I was told that TBS would screw it up and give you high background, as well as potentially interfere with your results.

I do not have a reference however I have heard it from several people. Perhaps it's an old-wives' tale?

I use PBS. I will look for phosphorylation state soon, so i'm interested in what you said. I also would like to ask if somebody has used anti phosphoserine antibodies to detect phosphorilated proteins. If so any recommendations? Is it also possible to quantify and compare phosphorylation state using this antibodies
Thanks

You have to remember that PBS is a phosphate-buffered saline and TBS a tris-base saline. Phospate groups in PBS can interfere with anti-phosphate antibody binding to your potentially phosphorylated protein and will bind all of the phosphate groups in the saline instead of your protein phosphorylation sites.
Also, milk is not used for blocking when anti-phospho antibodies are used due to phosphoproteins in the milk which will compete for the phosphorylation with your potentially phosphorylated proteins.

those are the same Westerns that we do. I was told that TBS would screw it up and give you high background, as well as potentially interfere with your results.

I do not have a reference however I have heard it from several people. Perhaps it's an old-wives' tale?

I use PBS. I will look for phosphorylation state soon, so i'm interested in what you said. I also would like to ask if somebody has used anti phosphoserine antibodies to detect phosphorilated proteins. If so any recommendations? Is it also possible to quantify and compare phosphorylation state using this antibodies
Thanks

You have to remember that PBS is a phosphate-buffered saline and TBS a tris-base saline. Phospate groups in PBS can interfere with anti-phosphate antibody binding to your potentially phosphorylated protein and will bind all of the phosphate groups in the saline instead of your protein phosphorylation sites.
Also, milk is not used for blocking when anti-phospho antibodies are used due to phosphoproteins in the milk which will compete for the phosphorylation with your potentially phosphorylated proteins.

the main difference is that TTBS contains the blocking agent Tween which increases specificity during Ab incubation whereas PBS has no blocker

if you really like to decide which buffer is to prefer you have to test both buffers for each Ab

one theory in perferring PBS for phospho-state analysis is that the high concentration of PO4/3-lowers the pressure of the hydrolization of phosphoryls from polypeptides

nevertheless, we have decided to use routinely TTBS although we analyze a lot of phospho-proteins


Purpose and function of blocking steps

The membrane supports used in western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding.

The proper choice of blocker for a given blot depends on the antigen itself and on the type of detection label used. For example, in applications where AP conjugates are used, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase. For true optimization of the blocking step for a particular immunoassay, empirical testing is essential. Many factors, including various protein-protein interactions unique to a given set of immunoassay reagents, can influence nonspecific binding. The most important parameter when selecting a blocker is the signal-to-noise ratio, measured as the signal obtained with a sample containing the target analyte, as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background staining and a reduced signal-to-noise ratio. Using excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the marker enzyme, again causing a reduction of the signal-to-noise ratio. When developing any new immunoassay, it is important to test several different blockers for the highest signal-to-noise ratio in the assay. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics.

Protein Detection Technical Handbook

This 84-page handbook provides a deep dive into the last step in the western blot workflow—protein detection. With a variety of detection techniques to choose from (chemiluminescence, fluorescence or chromogenic), you can select a technology to match your experimental requirements and the instruments you have available. Whether for quick visualization or precise quantitation, single-probe detection or multiplexing—Thermo Fisher Scientific offers a range of reagents and kits for western blot detection and subsequent analysis.


TBST [10X] (2 Citations)

A 10X Concentrated solution of Tris Buffered Saline with Tween® 20 with a concentration of 100mM Tris.HCl, 150mM NaCl, 0.5% Tween® 20 at pH7.5. The 1X Concentration is 10mM Tris.HCl, 15mM NaCl, 0.05% Tween® 20 at pH7.5. A dry buffer pack format is also avaialble to produce 1L of 1X TBST.

TBST is commonly used as a wash solution for Western blot membranes and microtiter plate wells in ELISA assays. The Tris Buffered Saline with Tween® 20 is an optimal formulation of pH stabilizers, salts and detergents designed to effectively remove excess material from membranes and microtiter plate wells without disrupting the antigen/antibody binding reaction. By maintaining the proper buffering environment, unbound components can be washed away without suppressing antigen-antibody binding interactions, thereby reducing nonspecific background and increasing the specific signal.


​Mild stripping

​15 g glycine
1 g SDS
10 mL Tween 20
Dissolve in 800 mL distilled water
Adjust pH to 2.2
Bring volume up to 1 L with distilled water

  1. Using a volume that will cover the membrane, incubate at room temperature for 5–10 min.
  2. Discard buffer
  3. Repeat incubation for 5–10 min with fresh stripping buffer
  4. Discard buffer
  5. Wash for 10 min in PBS
  6. Wash for 10 min in PBS
  7. Wash for 5 min in TBST
  8. Wash for 5 min in TBST
  9. Ready for blocking


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Western Blot Transfer Buffer Formulations

The standard transfer buffer for western blots, called Towbin buffer, is 25 mM Tris, 192 mM glycine, pH 8.3 &mdash usually with 20% methanol (vol/vol). Sometimes SDS is added to this buffer, generally in the range of 0.1 to 0.25%. This transfer buffer has both low ionic strength and low conductivity, which is optimal for tank (wet) blotting and for some semi-dry apparatuses.

Tris/glycine western blot buffer may not be suitable in some types of apparatuses for transfer of very high molecular weight proteins, which require lengthy transfer times. As transfer proceeds for an extended period of time, the production of heat decreases the resistance of standard western blot transfer buffer, causing the blot buffer to lose buffering capacity, thus reducing transfer efficiency. Additionally, the increased heat can cause gels to stick to the membrane, creating a handling problem for the soft, low-percentage acrylamide gels that are usually used for very high molecular weight proteins. Often, these gels must be carefully and laboriously scraped off the membrane.

For particular proteins, the choice of blot buffer can impact the efficacy of transfer. Generally acidic proteins are transferred more efficiently in a western blot buffer with a lower pH, and basic proteins are more efficiently transferred in a blot buffer with a higher pH. There are many buffers used for western blotting, such as the Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9) 10 mM CAPS, pH 11 and 10 mM CHES, pH 9.6.

For semi-dry western blotting, in addition to the standard Tris/glycine blot buffers, CAPS can be substituted for the glycine. A typical formulation has 60 mM Tris and 40 mM CAPS. An advantage of semi-dry blotting is that, unlike in tank blotting, the anode and cathode buffers are separated. This provides the ability to increase the efficiency of transfer by having different buffers at the anode and the cathode. For example, 15% methanol is generally added to the anode buffer, and 0.1% SDS is often added to the cathode buffer.


Use a final concentration of 0.1 - 0.2% Tween ® 20

Do not use SDS with Nitrocellulose membranes

Use a final concentration of 0.1 - 0.2% Tween ® 20

Do not add SDS to primary antibody dilution

Use a final concentration of 0.1 - 0.2% Tween ® 20

Do not use SDS with Nitrocellulose membranes

Use a final concentration of 0.1 - 0.2% Tween ® 20

Add 0.01-0.02% SDS final antibody dilution

Add 0.1% Tween ® 20 to wash solution

Do not use SDS with Nitrocellulose membranes

Add 0.1% Tween ® 20 to wash solution

Do not add SDS to wash solutions

For more tips on NIR Western blotting best practices, download Good Westerns Gone Bad.

No one blocking buffer works for every Western blot experiment.

Optimizing blocking buffer conditions will ensure you are getting the best Western blotting data. Don’t assume the blocker you used the last time is the right one for your current experiment.


Optimizing a Western Blot

So a bit of background, I'm a recently hired Research Assistant, and I'm still learning the ropes through the science world. There was recently some talk in my lab to get the iBlot or the Fast transfer western blot setup, so that we could get our Westerns done super fast.

But since those talks fizzled out, I was wondering if anyone had any advice on how to optimize a Western Blot for maximum efficiency. The key parameters I'm looking to work with here are :

Reducing background noise

So far, I've experimented with blocking in 5% milk, and also doing the Ab incubation in milk. That seemed to generally work way better than BSA. I'm also curious as to what the highest voltage you guys have run a transfer in is(for a regular transfer box.)

I've also heard rumors about drying out your transfer membrane before incubation, and not blocking at all. Seemed a bit weird, but I'm willing to try it out.

Any and all advice would be greatly appreciated. Thanks!

There's no one answer to perfect Western Blots. Every combination of antigen, antibody and samples has slightly different optimum condition. Depending on your goal - qualitative vs. semi-quantitative, order of magnitude of differences between samples, etc - these differences might be relevant or they might not be. Most peptide antigens can be decently detected with low fat milk powder as a blocking solution. Most. There are exemptions. And obviously detection of modifications - phosphorylation etc - cannot be done with milk powder. That's quite frankly why every third post in this subreddit is bitching and moaning about WB - they are the capricious little divas of molecular biology.

Like almost all techniques, doing a good immunoblot is a trade-off of time against quality. For my purposes, I always found that hand poured gels run at about 180 V for

60 minutes gave me the best resolution vs. time. I was looking for mobility shifts caused by phosphorylation and the resolution was usually pretty spot on. As r/OpthalmicObsessions already mentioned, each combination of target/antibody/samples is likely unique though so you'll need to think a little about the size of your protein and what you're looking for.

Regarding blocking, we always blocked at room temperature for in 5% non-fat milk powder dissolved in TBS with 0.1% TWEEN 20, so nothing revolutionary. 20 minutes blocking seemed to work just as well. Sometime Iɽ dry the membrane out and then block up to a couple of weeks later, but Iɽ always block.

Incubation with primary Ab was always overnight in 5% BSA in TBST, at 4°C. Generally, the way I see it is that for a consistently good blot, you can afford to spend a few hours running a gel and transferring it, followed by a decent blocking time and overnight incubation. Ultimately, a lot of the processes invovle are limited by physics so it's nearly impossible to speed them up without sacrificing at least some quality. All of the people I saw who cut corners or had special tricks to speed up blotting usually produced pretty poor blots and would have to go back and repeat for presentation/publication.

All this is a rambling way of saying that sometimes doing something relatively slowly can be the most efficient way, if it means not having to repeat the whole process.


Watch the video: Probe the Western blot for your target proteins using primary and secondary antibodies (January 2022).