Dear Lim,

Basically all it depends on the binding strength and specificity of tour primary antibody that you are using.

For those antibodies which are suspected to be more intensely/prominently binding, one can or advised to dilute it in 5% NFM (non-fatty milk) as it may prevent undue/promiscuous binding of the antibody, thus eliminating false positive signals in WB.

But there are antibodies which are very weakly binding, or their target protein may be scarcely present in the cell. For them it is always better and safe to use 5% BSA only, as because BSA is only one single type of purified protein and milk constitutes a number of mixed proteins which may hinder with the actual antibody binding.

Thus, using milk or BSA, will simply depend upon your primary antibody nature and of course on the availability of the taget protein of your interest.

But there is one occasion, where milk can never be used. Its in the case of diluting phospho-antibodies. As because the casein present in the milk may react with the antibody, thus debarring of the actual intended signal. One should always use BSA while diluting phospho-antibodies.

Dear Lim,

Basically all it depends on the binding strength and specificity of tour primary antibody that you are using.

For those antibodies which are suspected to be more intensely/prominently binding, one can or advised to dilute it in 5% NFM (non-fatty milk) as it may prevent undue/promiscuous binding of the antibody, thus eliminating false positive signals in WB.

But there are antibodies which are very weakly binding, or their target protein may be scarcely present in the cell. For them it is always better and safe to use 5% BSA only, as because BSA is only one single type of purified protein and milk constitutes a number of mixed proteins which may hinder with the actual antibody binding.

Thus, using milk or BSA, will simply depend upon your primary antibody nature and of course on the availability of the taget protein of your interest.

But there is one occasion, where milk can never be used. Its in the case of diluting phospho-antibodies. As because the casein present in the milk may react with the antibody, thus debarring of the actual intended signal. One should always use BSA while diluting phospho-antibodies.

if the primary antibody should be diluted in BSA, for blocking purpose, I should use BSA as well?

Yah, if you could do that, it would be better.

But again it is not absolutely mandatory I suppose, except when using phospho-antibodies and/or weak antibodies.

Skimmed milk: Pros: Much cheaper, blocks better. Cons: Cannot be used with Abs against phosphoproteins. Cannot be used with streptavidin-biotin-based systems. Lowers the sensitivity of some commercially available anti-His mAbs.

In general (and this is solely my experience, I cannot point you to an authoritative source on this) you can use BSA whenever the original protocol calls for non-fat dried milk (but do not be surprised if background increases). The same however, is not always true when the order is reversed.

We almost always block membranes in 5 % milk and test then if the antibody is working better in milk or in BSA. I agree that most of phospho-specitic antibodies work better when diluted in BSA (after milk blocking) but this is not true for all antibodies. Whenever I have to test a new antibody, I block in milk and then test both diluents for the antibody (if not specified). If I am satisfied with the result, that's it. If not, I test BSA blocking in a second step.

Usually, I use a mix of BSA and milk, but it is not recommended when using phospho-antibodies. For phospho, I recommend to use only BSA. Depending on the used antibodies, you have to try different blocking solutions and to play around with the % of added BSA/milk.

I always block my nitrocellulose membranes with 5% milk in TBST for 1 hour at RT and wash (3 x 5 mins). Then I put the primary antibody in 5% BSA overnight, wash (3 x 5 mins), incubate with secondary antibody in 5% milk for 2 hours at RT, wash (3 x 5 mins) and then detect. I routinely detect phosphorylated and total proteins, particularly using antibodies from Cell Signaling, and their recommended protocol for most of their anitbodies is to use 5% BSA to dilute. Using milk to dilute antibodies can be good if you need to reduce unwanted background, but it can also prevent antigen recognition by the primary antibody, resulting in a weaker signal. My rule of thumb is to always try the established protocol from the company, if stated. If that doesn't work, then I repeat trying different method (ie, if I used milk first and that didn't work, now I use BSA).

Hi Kee, I agree with previous answers. I used to use milk for both bloking and primary Ab steps and I used to have no background and good and clear bands. Check your antibody features and I would recommend you to do a WB using only milk and a second one using only BSA in order to compare them and know exactly which method works better for you.

I agree with all the previous answers regardin BSA, and non fat milk 5%, if you still have non specific signal you can also try to combine milk and BSA or use salmon sperme. You might also want to try different type of membrane (PVDF, Nitrocellulosis) as some antibodies give more background depending on the type of membrane (anti-Phospho PY20 antibodies give high background on PVDF for instance).

The best is of course to setup your antibody condition each time with different blocking solutions and membrane types.

Contrary to one of the commenter's experiences (and in line with Anne's answer), I use milk with all my commercial phospho-protein antibodies on a regular basis, and it works extremely well. Just make sure that you are doing overnight incubation at 4 degrees - it helps a lot. Minimal background and strong signal as a result (well - most of the time). Use milk throughout for better results - blocking, primary and secondary, as Miguel pointed out.

Thanks all of u

There was a similar discussion previously about the difference between BSA and non-fat milk for WB that you might find interesting:

https://www.researchgate.net/post/What_is_the_difference_between_BSA_and_Non-Fat_milk_NFM_blocking_of_the_membranes

I also use 5% skimmed milk in TBS as blocking buffer to prevent non-specific binding in western blotting, except phospho-antibodies. To detect phosphorylated protein, I use 3% BSA in TBS. The condition is good.

Which one of is better to use if we are using nitrocellulose and if we are using the PVDF. Does the blocking have an effect the type of membrane used?

Dear Tejas

When it comes to Western blotting, choosing the right membrane for your application usually spells the difference between success and failure. However, this is usually easier said than done since you need to consider the properties of your protein and the downstream detection steps required in your application before you can determine which membrane can give you the results that you need.

To do this, you need to take the membrane type, pore size and membrane format into consideration to help you choose which membrane to use.

Protein binding capacity. PVDF has a protein binding capacity of 170 to 200 μg/cm2 while nitrocellulose has a protein binding capacity of 80 to 100 μg/cm2. Since PVDF has a higher protein binding capacity, it also offers higher sensitivity. While this feature allows it to detect lowly expressed proteins, you are more likely to get higher background noise in your antibody detection steps when using this membrane. Nitrocellulose membrane, on the other hand, may not be capable of the detection sensitivity of PVDF membranes but it will produce a lower background noise.

Binding interactions. Protein molecules bind to nitrocellulose membranes through hydrophobic interactions while molecules bind to PVDF membranes through hydrophobic and dipole interactions. However, please note that nitrocellulose requires the use of methanol in the transfer buffer which may reduce the pore size of the gel and cause high molecular weight proteins to precipitate.

Physical characteristics. While nitrocellulose is brittle and fragile, PVDF is more durable and has higher chemical resistance making it ideal for reprobing and sequencing applications. Nitrocellulose can prove to be difficult to strip and reprobe without losing signal. However, supported versions of nitrocellulose membranes are considerably more durable and resilient than standard nitrocellulose membranes so they can be stripped, reprobed, and subjected to harsh chemical treatments.

Pore size. Both membranes come in typical pore sizes of 0.1, 0.2 or 0.45μm. The 0.45μm membrane is suitable for most protein blotting applications but for smaller peptides or lower molecular weight proteins (less than 15 kD), you should use 0.1 or 0.2μm pore size membrane. Note: When you are detecting a protein loaded at low levels or when quantification is considered critical, you should always choose the smaller size membrane.

Membrane format. There are several factors that you need to take into account when choosing the most suitable membrane format, including transfer system (semi-dry, wet or fast), convenience, price and flexibility. Pre-cut and pre-wetted membranes are the ideal choice when convenience, reproducibility and high throughput are of highest importance while rolls offers more flexibility since you can cut the membrane to the specific size of your gel. The only problem is that this can add extra time to your workflow and introduce variability in the membrane size. Pre-cut membranes, on the other hand, are available in a range of sizes suitable for all gel types. Using a precut membrane may result in better transfer reproducibility.

While both nitrocellulose and PVDF membranes are used for Western blotting and amino acid analysis, nitrocellulose is ideal in detecting low molecular weight proteins while PVDF is more suitable for detecting higher molecular weight proteins. In addition, nitrocellulose can be used for nucleic acid analysis and dot/slot blotting while PVDF can be used for protein sequencing and solid phase assay systems.

Keep in mind that you can get more reliable results from your Western blot experiment by choosing the correct membrane type. So, choose carefully and you will surely get the results that you need.

References:

http://blog.citeab.com/western-blotting-nitrocellulose-vs-polyvinylidene-difluoride-pvdf/

http://info.gbiosciences.com/blog/bid/203026/PVDF-or-Nitrocellulose-Which-Membrane-is-Best

Dear Alejandro Martin , 

Could you please explain more why we can not use skimmed milk for blocking my nitrocellulose membranes with streptavidin-biotin-based systems?

Thanks in advance.

It is a great thread, I got most from Anirban Chatterjee and Anne von Koschembahr's excellent answer. To help others sharing the same question with me, I cite these two experts' view as below:

Basically all it depends on the binding strength and specificity of tour primary antibody that you are using.

one occasion, milk can never be used. Its in the case of diluting phospho-antibodies. As because the casein present in the milk may react with the antibody, thus debarring of the actual intended signal. One should always use BSA while diluting phospho-antibodies.

Details:

I always block my nitrocellulose membranes with 5% milk in TBST for 1 hour at RT and wash (3 x 5 mins). Then I put the primary antibody in 5% BSA overnight, wash (3 x 5 mins), incubate with secondary antibody in 5% milk for 2 hours at RT, wash (3 x 5 mins) and then detect. I routinely detect phosphorylated and total proteins, particularly using antibodies from Cell Signaling, and their recommended protocol for most of their anitbodies is to use 5% BSA to dilute. Using milk to dilute antibodies can be good if you need to reduce unwanted background, but it can also prevent antigen recognition by the primary antibody, resulting in a weaker signal. My rule of thumb is to always try the established protocol from the company, if stated. If that doesn't work, then I repeat trying different method (ie, if I used milk first and that didn't work, now I use BSA).

J

Hi everyone,

To build on this question, I am wondering if a membrane that was previously blocked in BSA can be re-blocked in milk? Will that do any good?

Thanks!

Allyson