After cell lysis by bead beater, centrifuge at low speed (e.g. 5000 g) to remove unbroken cells and large fragments. Then centrifuge the supernatant at high speed (e.g. 100,000 g) to separate the membranes from the cytoplasm. Resuspend the membrane pellet in buffer. Mix some of the suspension with SDS-PAGE sample buffer to use for electrophoresis.

Adam B Shapiro

Thank you very much. To be clear, that protocol is intended for detergent free lysate?

If you want to have all the proteins in your western blot, just make a low speed centrifugation (5000 x g or 10.000 x g) and add some loading buffer, detergent is not needed.

If you want to have just your membrane proteins in your preparation, spin at 5.000 xg for like 10 minutes, and after that spin the supernatant at 100.000 x g for 1h, resuspend the pellet (without detergent) and just add it on the lanes.

However, be careful with your loading buffer, put the right amount of it and let it incubate for a little bit, so it can effectively solubilize your lipids, otherwise you may see some smears due to their presence.

You need detergent to dissolve the membrane proteins. There is SDS detergent in the loading buffer, so you don't have to add any other detergent. As Jorgaq Pata said, be careful not to exceed the capacity of the detergent in the loading buffer to dissolve the lipids, or else you will see a lot of smearing in the lanes. Basically, to get a good resolution gel, load no more membrane protein than is necessary to get the signal you need. The lipids will run to the bottom of the gel and cause some distortion, so choose a gel percentage that will keep you protein of interest in the top half of the gel.

Thank you.

For what duration should I carry out the low-speed and high-speed centrifuge steps?

It depends on the sample volume and length of the container. I would say that 10 min at 5000 g and 1 hour at 100,000 g are reasonable times.

Adam B Shapiro

Thank you very much. You seem to be very knowledgeable so I wanted to ask your opinion about one other thing:

I've heard from others on ResearchGate that boiling samples with transmembrane proteins doesn't work well for Westerns, as they're prone to forming aggregates.

In my case, I will want to detect multiple proteins, some of which are soluble and some of which are membrane. Is it okay to not boil all of them? I would think a high concentration of SDS will simply denature everything without need for boiling.

You could prepare duplicate samples, boil one and not the other.

You could also try using LDS sample buffer, with which you only heat to 70oC instead of boiling.

Adam B Shapiro

Thank you very much.

Yes, I was thinking I would probably have to do duplicates: +/- boiled.

I hadn't thought of LDS. I very much like that idea and will probably try it.

For the high speed centrifuge (~1 hour at ~100,000 g), I don't understand why the membrane proteins specifically precipitate while the other proteins do not. Why is that?

And if use too much detergent, is it possible they won't ever precipitate?

It isn't that the membrane proteins precipitate at 100,000 g, it is that the membranes containing them pellet. This only works if you do not dissolve the membranes with detergent first.

The 100,000 g membrane pellet can be resuspended in a small volume of buffer (one-tenth the original volume, for example) to allow you to do a protein assay on the pellet and the supernatant. That allows you to adjust the amount of protein run on the gel to be the same in all the lanes.

Ah. That makes sense. Thank you very much.

Should the buffer used to resuspend the membrane pellet contain detergent?

Gen Gi , it's better if the buffer has no detergent, mind that the solubilisation will start as soon as you add detergents, so you want to resuspend the pellet first and measure its protein concentration. This concentration is very important for the solubilisation, you must be sure that the quantity of the detergent you will be adding is enough to solubilize the membranes.

So, better resuspending your pellet in a buffer of choice, measure its concentration and if you want to work immediately, you may add the detergent after, otherwise conserve your resuspended pellet in a detergent free buffer.

Jorgaq Pata

Thank you very much.

So tests like BCA are accurate even if the proteins are not solubilized?

Why is it wrong to add detergent, measure, and then add more detergent if needed?

How can you determine if you've added sufficient detergent?

Well, for BCA, the theory is that lipids do interfere with the measurment. Practically in my case, this interference is very low, but you can try : carry on a BCA with and without SDS in your wells containing the sample.

You have to resuspend your pellet, resuspending with detergent is not practical because it will foam a lot. Secondly, you may want to keep your protein in membrane, unless you want to carry on solubilization in the same day as your membrane preparation (very time consuming).

So, to answer to the rest of the questions i need to know what applications will you do : just a western blot or you count doing some purification etc of any membrane protein?

If you just want a western blot, adding the Laemmli buffer (which contains 2% of SDS) is enough to allow the migration of membrane proteins. if you want to purify a protein, the question will be more complex.

It becomes complicated as I have multiple applications:

1. First I want to express in bacteria, and do westerns on WCL and membrane fractions

2. Later I want to purify recombinant forms from ecoli

I see. I was using this resource:

https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf

Which indicated detergents are pretty well tolerated, particularly if I dilute my sample.

" You have to resuspend your pellet, resuspending with detergent is not practical because it will foam a lot. Secondly, you may want to keep your protein in membrane, unless you want to carry on solubilization in the same day as your membrane preparation (very time consuming). "

I didn't realize this process was so arduous. What steps does solubilization entail? I would be happy to look it up myself, but the protocols I'm seeing don't go into adequate detail it appears

Okay, first you lyse your bacteria.

After that you carry on a low-speed centrifugation as previous suggestions said, you discard the pellet (debris) and you centrifuge the supernatant (1h, 100000 x g)

The supernatant of the ultracentrifugation will contain the cytosolic proteins, the pellet will contain membranes (including plasma membranes and thus membrane proteins)

Resuspend the pellet in a buffer, try it to homogeneize it as much as possible and keep it on ice.

Perform some assays, BCA to quantify proteins, SDS PAGE and Western blot. For these, you may take the volume you need from the sample and mix it with the Laemmli buffer, just do not overload it with proteins. Laemmli buffer has already SDS, so it will solubilize the membranes (keep in solution your proteins)

If you want to solubilize all of the membranes, adjust them to the needed concentration and add the desired detergent. Not all detergents will work, carry on small scale trials before deciding which is better to use.

After solubilization, centrifuge again at 100000 x g, 1H. The supernatant will contain membrane proteins that are being kept in solution by the detergents, the pellet contains insoluble membranes and aggregates.Work with the supernatant to carry on purifications etc.

Jorgaq Pata

Thank you very much for the detailed response. It is very helpful.

" For these, you may take the volume you need from the sample and mix it with the Laemmli buffer, just do not overload it with proteins. "

How do you determine if you overloaded the Laemmli buffer with proteins?

" Not all detergents will work, carry on small scale trials before deciding which is better to use. "

How do you determine whether the detergent was successful?

You can prepare several wells with for example 5 µg, 10 and 20 µg of proteins/well and see if the bands you get are nice or not. 20 µg may be a lot, so you 'll have to choose the one that looks better.

While doing small scale optimizations, you may have detergents that do not solubilize at all (your protein will be still in the pellet after ultracentrifugation), detergents that disrupt its activity even though they keep it in solution (probably SDS will do that or FC), and detergents that will keep it in solution and active. So you can control whether the protein is in the supernantant or not by SDS PAGE and do an activity assay to check if it is still active

Jorgaq Pata

Thank you again for the thorough and thoughtful answers.

What does solubilization entail after adding detergent?

Do you incubate it for a period of time? Do you mix it by pipetting? Do you assume the effect is near instantaneous if you homogenized the pellet before adding detergent? If solubilization fails with a detergent, how can I be sure it wasn't simply because I didn't put adequate effort into mixing it?

Add the detergent, put it at 4°C, let it for like 1h in over-to-end shaking. It will do it. The effect is very rapid, but let it 1h (or 2 in some cases).