Hi Silvia,

the blocking has two reasons. First the membrane itself has to be saturated with protein. Otherwise the antibodies would bind to the protein-loving membrane and you would get a very high and diffuse background. That is the main reason. Second reason is that the proteins in the blocking solution will also attach to the transfered proteins of your sample. They saturate unspecific binding sites to which the antibody, as any protein, could bind too. Unspecific protein-protein interaction is minimized this way and you will get more specific signals. The unspecific interactions are way less strong than the specific binding of an antibody (ab) to its target. So the ab will replace the unspecific proteins at the target. Each ab preferes a different blocking agent. Please check the manual of your ab to find the recommended blocking solution.

To the ladder signal:

This can have different reasons. If you are using fluorescence to detect your secondary antibody, a prestained ladder (mostly stained with Coomassie or other stains) can give signals in a fluorescent channel as well. Coomassie e.g. gives a very nice signal in a red fluorescent channel. If you use a ladder like the SPL-Calibrator (NHDyeAGNOSTICS) you will have a component that is cross-reactive to your secondary to give you a signal together with your target. This can be used to monitor the reactivity of you ab and to perform a blot-to-blot comparison. Maybe you can post a pic of your blot with some more information about your used protocol.

I hope I could help you.

Best,

Kristin

Hi Silvia,

the blocking has two reasons. First the membrane itself has to be saturated with protein. Otherwise the antibodies would bind to the protein-loving membrane and you would get a very high and diffuse background. That is the main reason. Second reason is that the proteins in the blocking solution will also attach to the transfered proteins of your sample. They saturate unspecific binding sites to which the antibody, as any protein, could bind too. Unspecific protein-protein interaction is minimized this way and you will get more specific signals. The unspecific interactions are way less strong than the specific binding of an antibody (ab) to its target. So the ab will replace the unspecific proteins at the target. Each ab preferes a different blocking agent. Please check the manual of your ab to find the recommended blocking solution.

To the ladder signal:

This can have different reasons. If you are using fluorescence to detect your secondary antibody, a prestained ladder (mostly stained with Coomassie or other stains) can give signals in a fluorescent channel as well. Coomassie e.g. gives a very nice signal in a red fluorescent channel. If you use a ladder like the SPL-Calibrator (NHDyeAGNOSTICS) you will have a component that is cross-reactive to your secondary to give you a signal together with your target. This can be used to monitor the reactivity of you ab and to perform a blot-to-blot comparison. Maybe you can post a pic of your blot with some more information about your used protocol.

I hope I could help you.

Best,

Kristin

Blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane non-specifically. Blocking is often made with 5% BSA or nonfat dried milk diluted in TBST to reduce the background.

Non-fat dried milk is often preferred as it is inexpensive and widely available. However, milk proteins are not compatible with all detection labels, so care must be taken to choose the appropriate blocking solution. For example, BSA blocking solutions are preferred with biotin and AP antibody labels, and anti-phosphoprotein antibodies, since milk contains casein, which is itself a phosphoprotein and biotin, thus interfering with the assay results. It is often a good strategy to incubate the primary antibody with BSA since it is usually needed in higher amounts than the secondary antibody. Putting it in BSA solution allows the antibody to be reused, if the blot does not give good result.

The concentration of the antibody depends on the instruction by the manufacturer. The antibody can be diluted in a wash buffer, such as PBS or TBST. Washing is very important as it minimized background and removes unbound antibody. However, the membrane should not be left to wash for a really long time, as it can also reduce the signal.

The following article will be so helpful

Article Western Blot: Technique, Theory and Trouble Shooting

Dear Silvia Caprari,

I fully agree with Shiva Kumar and Kristin Schulz. But I want to add a few things:

1.) it is important to use something for blocking which will not be detected by the later used antibodies, sera or whatsoever. Milk/Casein is most often used as it is cheap, but can get you in trouble when you use serum. BSA can also be detected if you use serum later, especially if you want to use human serum. Human albumin binds much less in that case, I compared it once with ELISA:

Article Quality of Recombinant Protein Determines the Amount of Auto...

In an extreme case of non-specific binding, you can also block the remaining reactive nitro groups of the membrane with chemicals (amino acids, ethanolamine) and use tween to block hydrophobic sides.

2.) Most people think that one antibody binds to one target molecule. But this is wrong, most antibodies bind to many similar structures. This is the natural design of an antibody, it shall react with similar things like similar viruses. Even if you use a good monoclonal antibody, you will still find a lot of off-target binding to many other proteins. Thats why we need usually two antibodies to be specific like in an sandwich ELISA, or need the additional feature of molecular weight in the western blot.

Ok. that also can mean that an antibody binds to albumin, but not to casein or vice versa. You need to check what is know about the one you want to use.

All those binding reactions are relative. The strength of its target protein binding usually correlates with the specifity of an antibody as a basic rule. An antibody might bind with low affinity to BSA, but with high affinity to your target protein.

So whether it works in your blot depends on the DILUTION of the antibody. if you would use this example antiboday dilute only 1:1000, you blot might be black. But if you dilute it 1:5000, you get only a binding to your target.

If you use an antibody the first time, I would try to optimize a few things first by the use of dot blots. those can be made very fast. You should dot your target protein, the lysate or your antibody and optimize things such as blocking agent and time, dilutions, substrate.

here I wrote a bit about substrates and show an (ugly) dot blot:

https://www.researchgate.net/post/Sensitivity_of_Western_Blots#view=5a221a7a615e27abec6ccbd2