Milk or BSA? Choosing a blocking protein for Western Blotting (WB)

Milk or BSA? Choosing a blocking protein for Western Blotting (WB) | CST Tech Tips

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6 жыл бұрын

How does choice of blocking protein - nonfat dry milk vs bovine serum albumen (BSA) - affect your Western Blot (WB) results?
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Transcript:
- What protein should I use to block a transfer membrane for a western blot? My name is Srikanth, I'm a product scientist at Cell Signaling Technology, and this is CST Tech Tips.
In regards to western blotting, a common question we always get is, what should I use, milk or BSA to block? So the purpose of blocking step is to reduce the amount of background due to non-specific bonding. Now BSA is only made up of one protein, BSA at 60 kDa, whereas milk is made up of many proteins, all of various sizes. So you get a much better chance to reduce more of the background banding.
We recommend that you use 5% milk in TBST, shaken for one hour at room temperature, to block all of our non-conjugate primary antibodies. This includes phospho-specific and total antibodies. Now I can already hear the clicking, comments, and hashtags, asking about "what about the phosphatases in milk?" Well, there are some papers out there that discourage you from using milk for phospho signal.
Let me address that by saying that in all of our in-house testings, we don't see any of these issues. CST scientists run so many westerns that we end up making milk once, at least once a day, sometimes multiple times a day. Now, if your milk buffer goes unused for a week, two weeks, even longer, that increases your chances of phosphatases affecting your signal. But again, we don't see any of these effects, because we use milk buffer fresh on a daily basis.
These are images of product number 13038, tested on lysates made from 3T3 cells treated with PDGF. The only difference between the blots is that one membrane is blocked in milk, and the other membrane is blocked in BSA. Clearly, the membrane blocked in BSA has a much higher background. Yet phospho signal is still strong and clean with the membrane blocked in milk.
So after the membrane has been blocked, please refer to the product's specific data sheet for the recommended antibody dilution buffer. It's either gonna be milk or BSA, depending on the specific antibody.
I hope this has been helpful. For full application specific protocols, they're available on cellsignal.com on specific product page. If you have any other questions, please feel free to contact any of the scientists at CST at cellsignal.com/support. For more CST Tech Tip videos, please subscribe to our KZfaq page. Good luck with your experiments. And we'll see you next time, thanks.
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Пікірлер: 12

@istavritmanyak 4 жыл бұрын

Hello, I have still some trouble understanding the mechanism of blocking since I am new to western blotting. I understand blocking buffer can fill protein-free empty areas on the membrane, but how does it allow my antibody to bind the target protein but not the other protein-filled areas? I mean how it does not cover my target protein if it blocks everywhere and does not block my interested protein antibody binding?

@cellsignaldotcom 4 жыл бұрын

Thanks for the question, Basak. When thinking about the interactions of your antibody and blocking proteins with proteins on your blot, consider the relative affinities (KD) and off-rate (K-off). The interaction of the antibody and its antigen (target protein epitope) should be high affinity and have a low off-rate, while the interaction of an antibody and off-target peptides, as well as that of blocking protein to proteins on your blot, should be low-affinity and high K-off. In the latter case, the blocking proteins (or antibody) are constantly binding and unbinding and exchanging off the membrane, which allows a higher concentration of blocking proteins to reduce off-target binding of the antibody by mass action. Switching our focus again on the antibody target epitope, there's nothing that would prevent the blocking proteins from binding here. But again, this is a low affinity interaction with high K-off, so the blocking proteins will exchange on and off the target protein, giving the antibody an opportunity to access its specific epitope. Once bound, the high KD and low K-off of the antibody-antigen interaction mean that the antibody will stay tightly bound during incubation and subsequent washes, allowing you to detect your target protein.

@sudarrbm 4 жыл бұрын

Hello, If the issue is choosing the correct blocking buffer, (in the example that is given ) why is it that there are non-specific bands only in the protein lane when immunoblotting and not just a darker membrane in the background ? Shouldn't the proteins in the blocking buffer bind to only empty spots on the membrane and not affect the areas where proteins are already bound ?

@cellsignaldotcom 4 жыл бұрын

Thanks for the question. The blocking proteins are absorbed onto the membrane including both "empty" areas without protein, and to the lanes where the sample proteins have been transferred. Ideally, blocking proteins will A) prevent non-specific absorption of the primary/secondary antibodies to the membrane, and B) reduce low-affinity binding to off-target proteins in the lanes. Please note that the example shows a comparison of blocking with milk vs. BSA, not milk vs. no blocking. BSA and milk both accomplish A), but milk does a better job at B).

@sudarrbm 4 жыл бұрын

@@cellsignaldotcom Thank you very much !

@simon6932 3 жыл бұрын

Do you see any advantage to using commercial blocking buffers such as Bio-Rad EveryBlot Blocking Buffer or LI-COR Intercept blocking buffer? They're supposed to have less impurities than milk.

@cellsignaldotcom 3 жыл бұрын

Thanks for the question Simon. As the video mentions, our scientists routinely use fresh milk buffer for non-conjugate primary antibodies, and we get robust results. I believe for the majority of those western blots, HRP-based chemiluminescent detection is being used with a digital imager. If you have concerns about a particular detection method or a particular CST antibody, feel free to contact a scientist at cellsignal.com/support and they may be able to provide more insight.

@simon6932 3 жыл бұрын

@@cellsignaldotcom I don't have any concerns, I'm just wondering whether a commercial blocking buffer might make my results even cleaner than they already are with milk.

@mateenkhan1990 5 жыл бұрын

Hi Sir. Can you please recommend me a buffer which is good for the extraction of different milk proteins... And also what should be the pH of that buffer? I will wait for your reply Sir. 🙏

@cellsignaldotcom 5 жыл бұрын

Hi, our workflows and protocols are generally starting with biological materials such as cells and tissues; milk is used in the Western Blot protocol as a blocking reagent rather than a sample. If you will be performing Western Blot or other antibody-based assays and have a question about those protocols, you can contact one of our scientists at www.cellsignal.com/support.

@SCSCS 5 жыл бұрын

Why would using a week long milk buffer increase phosphatase activity?

@cellsignaldotcom 5 жыл бұрын

Good question! Here is a reply from Srikanth: Integrity of the milk buffer becomes compromised as time goes on from repeated use. In general, enzymes in milk come from numerous sources including but not limited to the native milk, bacterial contamination from opening and closing the bottle, or in somatic cells present in milk. Milk contains casein, which is a family of phosphoproteins (and phosphatase substrates). The proteins can be degraded by enzyme action and by exposure to light. Additionally, agitation brings enzymes (such as lipases, proteases, phosphatases, etc.) into contact with the milk proteins resulting in degradation. Phosphatases are very stable which allows them to outlast their substrates; with time, phosphatase activity remains the same but there are fewer non-specific (i.e., competitive) substrates left in the milk. Then, the phosphatase activity is more likely to affect your phosphoprotein of interest. Further reading: www.ncbi.nlm.nih.gov/pmc/articles/PMC4887191/ www.ncbi.nlm.nih.gov/pubmed/2434996 Farkye, N. Y. Other Enzymes, in: Advanced Dairy Chemistry, Vol. 1 Proteins. 2003, 3rd Ed. Fox, P. F., and P. L. H. McSweeney, eds. Kluwer Academic/Plenum Publ., NY.