Dear Ravinder,

Milk is cheaper! When you are not blotting for phosphorylated proteins it is OK. Milk is a mix of different proteins whereas BSA is just one. When blotting for phosphorylated peptides there can be binding to some phosphorylated milk proteins instead of your target, with BSA this does not happen. During all other cases milk is ok.

Dear Ravinder,

Milk is cheaper! When you are not blotting for phosphorylated proteins it is OK. Milk is a mix of different proteins whereas BSA is just one. When blotting for phosphorylated peptides there can be binding to some phosphorylated milk proteins instead of your target, with BSA this does not happen. During all other cases milk is ok.

Hi Leena,

Thank you for your information

Is there any concentration difference effect of blocking agent between milk and BSA, because we are using both at same concentration (5%)?

Hi Tomas

Thank you for your information

As others said, milk is cheaper and is as effective as BSA (or even more) for blocking in the majority of the cases.

Some time ago when I was working with antibody preparation i had this weird Western where the entire membrane was purple except the regions of the lanes with the bands. After some research I found that antibodies from the preparation were reacting against the milk proteins. This effect is called 'negative western-blot' (don't know if its a official name or just a nick name).

Although it might be uncommon, reactions like that might occur and you will need to use a different blocking reagent, so don't think in terms of "why user another one if milk is cheaper", think about "which one is more adequate to my situation".

Often it makes no difference if you use 5% BSA or nonfat dry milk for your primary antibody. However, you should incubate the primary antibody at the recommended dilution with the recommended blocking agent (either 5% BSA or 5% nonfat dry milk) as descibed by the manufacturer. Beyond that, some manufacturers (e.g. CST) highly recommand that you to use 5% nonfat dry milk in TBST as blocking agent for the secondary antibody, since BSA-based blocking solution results in higher levels of background bands.

While I agree with comments above, it is worth noting that results are empirical, by which I mean different antibodies yield different levels of background and the best solution if you are having issues with background or sensitivities is to try several different blocking buffers. Blocking definitely can effect sensitivity of your immunoblot.

Milk is cheaper, and usually a better blocking agent than BSA, but this is somewhat antigen-dependent. In my experience, some antibodies only work well with BSA.

yeah cheaper!!! and some ab crosslink with BSA getting like dirty your western when you take the film, other ab work better with BSA...I used 5% milk on TBS-T 1h and if i have no time 10% 30 min...and it works :P

Hi Ravinder,

I'll tell you a secret: Most labs have developed a "traditional" standard protocol including the use of milk or BSA! But in my experience this isn't a good idea, since usually it sufficient to simply block your membranes with TBS-T over night @4°C (or >1h RT). It worked the best for me in 95% of the cases. I only use proteins for blocking in case TBS-T is not working well! Blocking with milk or BSA often leads to a decrease in signal in my experience.

Best Kai

Hi Kai,

If we use alone TBS-T for blocking, will it work?. What is the mechanism of TBS-T as a blocking agent. Since we are using BSA or Skimmed as a blocking agent? Please can you explain the mechanism of these agents as a blocking agents?

Hi Ravinder,

The only difference is that milk is much cheaper compared to BSA. Definitively, you can use BSA in your western blot.

My opinion is also in alignment with all the comments already made addressing the issue whether 5 % milk in TBS-T is better blocking agent over 5 % BSA in TBS-T for western blot.

Just as an additional remark, the selection of blocking buffer has to be customized depending upon protein of interest (phosphorylated vs non-phosphorylated) as well as the end point detection system. Even 5 % BSA in TBS-T may not be suitable for non-phosphorylated protein for a Li-Cor Odyssey IR based detection system. That's why Li-Cor is selling a proprietary formulation of blocking buffer, which is very much expensive (Odyssey™ Blocking Buffer, 927-40000 series) but gives a very clean data. We have been using this for long time.

We are now using a fish protein based commercially available blocking buffer (AquaBlock™/EIA/WB, East Coast Bio, Cat# PP82) with almost half of the price of Odyssey™ Blocking Buffer and we are very much satisfied with the performance.

I am sharing a combined file for the data sheets for these two blocking buffers.

Alternatively, Gelatin blocking. I have used this for carbohydrate detection.

Hi Ravinder,

the Tween will saturate the free binding sites I guess. Just give it a try. As I already said it worked well for me in most cases.

Best,

Kai

Hi,

Mainly the price, I usually used milk to saturate membrane it worked fine with protein that I studied.

Best,

Cyrille

In many cases for virus infections diagnostics we used successfully blocking with 0.5-2% Tween-20.

Here are some information about blocking buffer.

Personally, in most cases, I use 5% skim milk but when I use biotinylated antibodies and streptavin conjugated antibodies, I use 3% BSA...

I think milk is significant source of biotin... isn't it?

Best,

Hyun

casein is probably what you're thinking of. A mixture of several phosphoprotein found in milk. Most ICC kits from companies, such as Invitrogen or Roche provide a "proprietary" powdered mix that mainly consists of casein. You just mix in buffer to a percentage that works for your system and use it for pre-block/block.

We have used both skimmed milk powder and BSA, and either one work fine for most Westerns. As mentioned by others, milk powder is much cheaper than BSA, and is hence preferred.

I agree with the reasoning of M. Rahman. SMP is much much much cheaper as compared to BSA. So it is the first choice for blocking for many. Moreover, generally BSA and SMP give similar results; many users say this and I have also used SMP in few blots with satisfactory results. However, BSA is clean to use and is preferable if one can afford except for specific cases like if one is looking for phosphoproteins.

Now I must muddy the water. All of the this advice is good advice, but should never be applied to every situation. We use a phosphotyrosine 1068 antibody that needs milk for blocking, otherwise there is high background and unclean banding. While it is well known that there are proteins and such in milk that could interfere with phoshotyrosine antibody, it is not an always situation to use BSA with pY. It is however a good rule to try BSA first with pY and if that fails to meet expectations, do not fear trying to block in milk.

Good information Jamie.

In most cases milk will be ok. From my own experience with blotting ADH and ALDH enzymes it was even better than BSA (background was lower, specific bands were clearer).

Milk is much cheaper than BSA, you don't have to use milk powder specially selled for laboratory use, you can use milk powder selled in the super market - without any difference in the quality of your results.

If you are not using antibodies against phosphorylated proteins, use milk for all of the reasons mentioned above. If the antibody is not specific against any consensus group use BSA, but If the antibody is specific against a consensus group use first BSA and if the results do not meet your expectations try milk.

First choice as a blocking agent is skimmed milk powder and second choice is BSA. This appears to be the consensus. This is the general choice for ELISA and immunoblots; exceptions would always be there. Isn't it?

That's what.

I used skimmed milk (5%) and got very good result.

The best blocking agent is BSA but Milk is easier to get and handle and least but not the last advantage is that it is more economical to use.

when i mentioned consensus I ment antibidies against for example antibodies against PKA consensus.

Milk is cheaper and work most of time. However, fat free milk also contains some immunoglobulin that can be (some time, depending on how much they are) recognized by the secondary antibody yielding huge background. I have experienced this mainly when my primary is made in goat and I use anti-goat as secondary.

If this happens, use BSA (ultrapure, immunoglobulin free). Fish skin gelatin is another option.

Another way is to block with Tween; we did this and it works:

Block the blot in 0.5 % Tween 20 for 30 min at RT. DO NOT GO LONGER as protein bands may be lost from the blot.

I have tried a lot of different blocking reagents over the years and agree that in general powdered milk is a reasonable, cheap starting point for many antibodies. However, it is important to keep in mind that the purpose of a blocking reagent is to bind to "non-specific" sites on the filter (be it nitrocellulose or PVDF or other) to lower background overall. And in the process most blocking reagents also can effect the sensitivity or strength of the specific signal. It is a bit of a trade off. So if you are going to use one antibody a lot and if quantitation is important, I encourage you to try several different blockers and different concentrations of those that you are considering using. Nothing beats empiricism!