This is a bit of a definition issue. Non-reducing does indeed usually mean that only the b-mercaptoethanol is omitted from the sample buffer. As SDS is still present, the PAGE will still be denaturing. However, you might actually want to try a native PAGE as well(no b-mercaptoethanol and no SDS either) to see the difference. So you have 4 options altogether:

1. Standard SDS-PAGE (reducing (b-merc) and denaturing (SDS))

2. Non-reducing (no b-merc, but still SDS)

3. Non-denaturing (no SDS, but still b-merc)

4. Native (no b-merc and no SDS)

Might be worthwhile to consider all options to see if either disulphide bridges or protein folding play a bigger part. Up to you.

This is a bit of a definition issue. Non-reducing does indeed usually mean that only the b-mercaptoethanol is omitted from the sample buffer. As SDS is still present, the PAGE will still be denaturing. However, you might actually want to try a native PAGE as well(no b-mercaptoethanol and no SDS either) to see the difference. So you have 4 options altogether:

1. Standard SDS-PAGE (reducing (b-merc) and denaturing (SDS))

2. Non-reducing (no b-merc, but still SDS)

3. Non-denaturing (no SDS, but still b-merc)

4. Native (no b-merc and no SDS)

Might be worthwhile to consider all options to see if either disulphide bridges or protein folding play a bigger part. Up to you.

You are right. just exclude DTT / Beta mercapto ethanol.

Thank u very much! Now I' m thinking about an other question: If I decide to have a native wb, do I have to heat the samples?

You are right. Remove 2-mercaptoethanol in sample buffer. For native WB do not heat the samples.

Removing 2-mercaptoethanol and SDS is an obvious solution to eliminate denaturing conditions.

However, certain critical issues to consider for non reducing western blots are:

1. Net charge of protein of interest.

2. Thermostability.

3. Protein size.

4. Transfer conditions.

1. Net charge:

Is your protein positive or negative charged? Using reducing conditions would eliminate the charge bias as they confer uniform negative charge on the peptide and the migration is mass dependent towards the positive electrode. So check out this parameter for your protein.

2. Thermostability:

For sample heating, check the level of thermostability of your protein. If your protein of interest is very thermostable then heating can help denature your background proteins and still maintain the native nature of your protein. Heat is also generated when running the gel. Take appropriate measures. Your western blot will work if your antibody recognizes a very specific surface epitope. You might run into a problem of multiple background bands due to cross reactivity to other denatured proteins.

3. Protein size:

Use SDS in transfer buffer when protein size greater than 100kDa. Don't use when protein size less than 100kDa. Yet again here, the question of denaturing conditions still persists.

4. Transfer conditions:

Heat is generated during protein transfer. A wet transfer in cold conditions might help.

Low transfer times (15-20 minutes) are better for smaller proteins (< 30kDa).

These are certain issues I could think of. You need to take appropriate measures and modify your protocol accordingly depending on the nature of your protein.

I would ask u something more about WB. What does it happen if I don't heat the sample, I remove b-mercaptoehtanol and SDS from the sample buffer but I mantain SDS in electrode and blotting buffer?

Check page 6 in the following link regarding non-reducing and native samples.

For SDS requirements in blotting buffer, go to page 10.

http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf

P.S. I do not endorse nor intend to promote any particular commercial provider here.

Only just a comment.

beware if you would analyze monoclonal antibodies, you will find the whole antibody with 2 heavy (H) and two light (L) chains. Hybridoma cells producing often some nonsense: H2L1, H2, L2, H1, L1. H1L1. All fragments containing at least one L and one H-chain can react with your antigen. But all of this fragment will become obvious at the expectable molecular weights. That's something you would never see under reducing conditions.

Many SDS-EP were made by comparing within one gel the reduced and the non-reduced version.

PS: Heating is only to increase reaction speed for the reduction. This will happen anyhow, but slowlier. So you can have an incomplete reduction

Hey Guys,

I need to run Collagen 1 alpha western blot. I usually prepare my samples with loading buffer + DTT + Heating, but western blot just wouldn't work...

Some say I need to use Non-Reducing Denaturating western blot with this specific antibody... For this, I will skip DTT when I am preparing my samples but still heat them, right?

What I am wondering is what happens to my TATA or HSP90 that I use as controls?

Hi, Fahrettin,

If you look for non-reducing conditions, u should eliminate the DTT, but also beta mercaptoethanol, which is included usually in some commercially available loading buffers.

Denaturating implies addition of SDS to loading buffers, gels and electrophoresis buffers and also heating.

I have no experience with TATA of HSP90, sorry. I would load both a reduced and a non-reduced sample and probe both for your loading controls, to see if there is any change.

Hi Fahrettin,

When you say your blot isn't working do you mean you cannot see any bands or do you see bands that are different molecular weights? 

Collagen 1a1 is a large molecule, so make sure that your transfer conditions are working well. When I did collagen WBs I found that a semi-dry transfer was too inefficient and ended up switching to tank transfer with extra SDS in the transfer buffer, which worked much better.

I didn't have any problems detecting denatured, reduced collagen 1 with an anti Col1a1 antibody I purchased from Abcam. I'm happy to give you specifics if you're interested.

Dear Kerstien,

Thank you for your feedback. I couldn't get any bands at all at the beginning. I prepared my samples without adding DTT and avoided heat treatment (basically denaturated but non-reduced [I still used SDS]); then it worked! I had quite nice bands.

Funny thing is protein results were different than transcript results. I repeated WB 3 times just to be sure and yes, mRNA and protein levels were different. I am planning to do some IHC now.

Couple of things, addition of BME does a very specific kind of denaturing, i.e. reducing disulfide bonds. SDS basically elimnates all hydrophobic and non-covalent linkages (aside from very special cases like detegent resistant inclusions). So, basically when you add BME and reduce a protein, this will open up the proteins conformation completely, and increase drag, thus slowing down the protein, this very apparent in some cases, in other cases, addition of a cys modifier like mPEG or something else which would add a fixed amount of wieght per cys would make the reduced and non-reduced forms distinguishable on a conventional SDS PAGE (with no added reducing agents). Another important note, sometimes, your protein may be complexed to gian homo/hetero-oligomers which may not enter a 10% gel, or just run wierdly- this isnt common, but I suppose it may happen from time to time.

EDIT: DTT is also a reducing agent