My thoughts:

"There are a few questions I want to know:

1. The rationale of drying the membrane in storage,"

### In my opinion, there is no real rationale for drying the membrane after probing. Air drying it BEFORE probing really helps "fix" proteins to membrane and can give higher signals since less protein is lost during washing steps etc. Indeed, if it is PVDF and the membrane is dry, you will have to first re-wet it for 10secs in METHANOL and then water/PBS to really get it saturated in aqueous buffer. Whether you dry before or after. Actually, a 20% methanol solution with also work.

Also, IF YOU HAVE ADDED ANTIBODIES ONCE, YOU MUST STRIP IT, before drying it since the antibodies will attach almost permanently when dried.

"2. The consequence of drying the membrane, why the membrane should be kept wet?"

### Dry only after stripping. A wet membrane is much easier to re-wet with PBS/aqueous solutions. Generally, re-wetting a dry membrane with Methanol is OK, but some protein loss may occur as both membrane and Methanol compete for hydrophobic sites. So try 20% methanol and then 100% Methanol.

"3. How long can a membrane be kept, without affecting the result?"

### Months.

4. Which method can store the membrane for a longer time?

### Wet membranes are fine to store if you keep them relatively flat.

SO,

1. After transfer air-dry the membrane for about 30-45 mins. If you are short on time, then forget this.

2. Re-wet the membrane with 20% methanol in PBS or just your transfer buffer. Wash with TBS.

3. Probe membrane as usual,.

4. After probing, STRIP MEMBRANE with you favorite solution.

5. NEUTRALIZE/WASH with TBS containing 0.05% Azide. Don't worry this azide will get washed off when you block and wash before HRP step.

6. Wrap membrane in Saran wrap - 2 rounds. One round face up, one round facing down.

7. Place membrane in a good ziplock bag. ADD 2-3 mls of water (DH20) + 0.05% azide, and remove air from ziplock and immediately zip it up trying to keep air out, and water in !! Store at 4C. The water will prevent drying as long as zip is good !!

8. For use, remove membrane, wash briefly and then block.

Good luck !!

Hi,

you can either dry the membrane or froze it at -20C. It doesn't seem to affect further detection.

Personally, I prefer to freeze my membrane and I know that I can re-use them within 6 month.

Good luck

I freeze my membranes at -20..and works good

before blooting at room temp, dry!

after blooting I keep the results in a zip bag at 4ºc in the dark and wet....

I'm used to keep it wet on TBST at 4°C, and I still have a good signal after 2 weeks, without background. I have never tried to store it longer than that. Anyways, I don't know why anybody would keep a membrane for longer than 1 month... It would be better to run another gel if you want to be sure that all the protein is still there.

Keep at 4 degree in PBS. If u dry it completely it might become fragile. So we need to keep it wet.

First of all, for long term storage you have to dry the membrane. If you just want to keep it overnight put in TBS-T in the fridge.

For long term storage, strip the membrane of any antibodies and blocking agents (e.g. stripping buffer: 25mM Glycine, pH 2.0, 1%SDS). After stripping, wash the membrane in TBS-T, then de-activate in methanol and air dry till completely opaque. The dry membrane you can either wrap in Saran wrap and put at +4degC or put on a piece of cardboard, wrap in foil and store at -80degC. Be careful not to fold your membrane, creases on your membrane will show ugly marks during detection.

1. bacteria can eventually grow on a wet membrane, also it's not very practical. How many blotting boxes can you keep in your fridge? :)

2. If you dry the membrane before you strip it, all the antibodies will bind to it permanently.

3. Like people above said, up to 6 months or maybe even longer.

4. If you keep it dry at -80degC, it will allow you to store them the longest possible time.

Methanol + dry and -80 and you can store it up to 6-12 months. This is the method recommended by Millipore.

agree with most of the above.

Personally, I prefer to blot on the same day. however, I also keep the membranes in the blocking buffer at -20.

For lab use, storage of the blocked membrane at -20 °C between two sheet of filter paper is the best. You have to avoid the contacts with detergents. This molecules will interact slowly with the hydrophobic interactions between your proteins and the NC or PVDF. This conditions will give you after a short time a dry membrane. This will keep your proteins for 1 - 2 years alive.

For longer storage a stabilization with sugars are helpful. in commercial tests either glucose (1-3 %) or mannose is often used (or a combination of both). Also pure proteins like Albumin or hydrolyzed gelatin are often used in the last blocking step. It's much better defined (and free of any proteases) as skimmed milk. Dry the membrane again in the freezer between two papers. Seal then in a air prove bag again between two sheets of filter paper. Put an dry bag on top.

This package can be stored for several years at room temp.

Usually I prefer keep the membrane in blocking buffer at +4 °C , but sometimes I dry it for some weeks and I had good results.

Whenever you are storing the membrane under liquid conditions you have to avoid bacterial contaminations. From a physicochemical point, you will have always an replacement (due to the excess of the blocking protein) of your proteins from the electrophoresis. Just compare the lower detection limits for some specifically stained proteins. It's not that much and depends on the hydrophobicity of your protein.

I agree with others that the things are good with PVDF membrane, if you dry after activating it with Methanol, and store it for longer at +4. It would be sufficient to store the membranes at +4, while -20 and -80 degrees are only needed if you think to store for years.

In my lab, people have also used nitrocellulose membrane (no need to activate with Methanol), some had stored the membranes at +4 degrees, and some at room temp. I came to know that both worked without problems after 6 months.

It would be also useful to know about other's experiences with nitrocellulose membranes.

agree with most of the above.

the membrane should not be dried in any process,Keep at 4 degree in PBS. If u dry it completely it might become fragile. So we need to keep it wet.

Dont dry membrane, just sandwich membrane with filter paper wet with TBS-T and store for short time at 4 degree

I agree with most of the above, and I agree not to dry the membrane.

My questios is , what could be the cause of loosing the signal from the membrane if not drying? I use the membrane maximal 2 times, that means I prob it with a 1ry antibody and get a good signal, next day , another antibody and still get a good signal. Unfortunately , afterthat I do not get any signals from any other antibodies even if I went back and tried the same antibodies.

is it the washings?

You can keep your membrain in non fat dry milk blocking buffer or albumin blocking buffer at -20C for lung time.

Hani, I also think it is due to the prolonged washings. I have also observed that the signal is either partially or completely lost, after washing the membrane for 2 days with PBS Triton. One way to be sure if there is no problem with the proteins (on the membrane), is to check with Ponceau red.

... But issue with Ponceau red is, that you can not do it anylonger after you blocked with BSA or milk already because it will just show you all in red since your BSA or milk did contain so many proteins that your whole membrane will be positive stained.

Store the membrane between two sheet of filter paper frozen (after blocking, but always without any detergents). As mentioned before, the hydrophobic part of the detergents will go slowly (but surely) between your protein and the membrane.

The same reason why you should not store the membrane wet in the blocking buffer.

Only if you are planning a long time storage, you have to add some sugars (1 ... 4% glucose and or mannose). This will keep a minimal amount of water with the proteins to avoid the denaturation on membrane. Then you can store the strips or the membrane even at 4 °. But seal then in a bag to keep the conditions as constant as possible. Avoid higher temperatures for longer times. That's what is done in the diagnostic industry.

If you are interested in more details, run experiments (with dropped proteins on a membrane) to compare the different blocking and storage conditions. Use serial dilutions of your antibody and look for the lower detection limit. You will see that whenever a wet storage, that your detection limit is fading away.

Store the membrane in 5% nonfat milk TBST for 2 weeks

Why milk? If you do not add 0.5% Sodium Azid into the milk, the milk will be bad after two weeks (bacteria contamination). If you want to store it wet, TBST itself is fine. But usually, most of the people either freeze the membran being a bit wet and covered in Cling wrap OR store it dry covered in Cling wrap at 4degrees.

When you add Sodium Azid into the buffer, the membrane will keep for long time. But the Sodium Azid is a cancinogen, you should be very careful to handle everything.

The membrane always be stored in closed bag with 5% nonfat milk TBST in 4 degree

Be careful. NaN3 is a very potent inhibitor of the peroxidase. Make sure that you are changing the reservoirs for the different incubation steps.

We usually store the PVDF and nitro membrane in clingfilm @ 4°C for more than one year. Recently I re-blotted a membrane prepare in the 2010 and the results have been ok.

Sodium Azid is also a toxic compound like cyanide, so be very careful.

At the end of the transfer we usually put the membrane in Methanol for few seconds, let it dry under the hood and keep it at RT for days. We never kept it for very long period of time, so we don't know.

all suggestion are OK

You can keep it in bag with blocking buffer or PBS in -70 for long time.

First I strip it and then I store it in TBS-T at 4C. As long as you keep it moist at cold ( prevent bacteria growth) it is ok. My TBS-T has BSA 1%.

We have stored the membrane in TBS-T at 4C for up to two weeks without any ill effects. However, storing in just plain TBS for more than 2 weeks will prevent some erosion of protein that would have otherwise occurred using Tween 20.

You can store your Western Blot membranes for long time in 5% non-fat dry milk or 1% BSA in hybrid bag at -20C, it will work well and you will have good results.

My thoughts:

"There are a few questions I want to know:

1. The rationale of drying the membrane in storage,"

### In my opinion, there is no real rationale for drying the membrane after probing. Air drying it BEFORE probing really helps "fix" proteins to membrane and can give higher signals since less protein is lost during washing steps etc. Indeed, if it is PVDF and the membrane is dry, you will have to first re-wet it for 10secs in METHANOL and then water/PBS to really get it saturated in aqueous buffer. Whether you dry before or after. Actually, a 20% methanol solution with also work.

Also, IF YOU HAVE ADDED ANTIBODIES ONCE, YOU MUST STRIP IT, before drying it since the antibodies will attach almost permanently when dried.

"2. The consequence of drying the membrane, why the membrane should be kept wet?"

### Dry only after stripping. A wet membrane is much easier to re-wet with PBS/aqueous solutions. Generally, re-wetting a dry membrane with Methanol is OK, but some protein loss may occur as both membrane and Methanol compete for hydrophobic sites. So try 20% methanol and then 100% Methanol.

"3. How long can a membrane be kept, without affecting the result?"

### Months.

4. Which method can store the membrane for a longer time?

### Wet membranes are fine to store if you keep them relatively flat.

SO,

1. After transfer air-dry the membrane for about 30-45 mins. If you are short on time, then forget this.

2. Re-wet the membrane with 20% methanol in PBS or just your transfer buffer. Wash with TBS.

3. Probe membrane as usual,.

4. After probing, STRIP MEMBRANE with you favorite solution.

5. NEUTRALIZE/WASH with TBS containing 0.05% Azide. Don't worry this azide will get washed off when you block and wash before HRP step.

6. Wrap membrane in Saran wrap - 2 rounds. One round face up, one round facing down.

7. Place membrane in a good ziplock bag. ADD 2-3 mls of water (DH20) + 0.05% azide, and remove air from ziplock and immediately zip it up trying to keep air out, and water in !! Store at 4C. The water will prevent drying as long as zip is good !!

8. For use, remove membrane, wash briefly and then block.

Good luck !!

Does dry-air storge work for nitrocellulose membranes?

I would always keep the membrane wet in TBS-T. Store it in a plastic bag wit TBS-T, in fridge at 4 degrees, on dark.

For nitrocellulose membranes (i have better experinces with those than with nylon or PVDF, maybe too complex for me :-p ):

After transfer, I block the nitrocellulose membranes carefully:

means 5% dry milk powder, 0.2% tween-20 plus TBS pH 7.7 as TRIS blocks too, 5 mM EDTA (In my opinion very important to prevent oxidation and microbes too, will also block), NO AZIDE; I use 0.01% thiomersal

In some cases, I change from milk powder to 2% human albumin from clinically used infusion bottles (which in my opinion is best for any work with human serum but also with very senstive biotin application as milk powder might interfere with this) or special purfied BSA at 0.5-2% (for certain fluorescence games). I never use commercial buffers like casein block buffers due to bad experiences after thawing.

2h will do, but usually do it overnight.

For storage I just reemerge the membranes after development with ECL etc. directly in blocking buffer for a few minutes and freeze them wet in a bag at -80.

Sometimes I store the membranes just after staining, with HRP, with ECL or whatsoever directly to -80, no block buffer. But this only for storage of a few days. Or I freeze after blocking.

Always in a still wet form (add block buffer, EDTA is important) in a small trash bag, no paper. I don't mind of any antibodies, reagents, whatsoever.

Ok. The bad side: The nitrocellulose membranes are very very fragile and break easily at -80. Especially with the modern fast blot methods which destroy a bit of the membrane due to heat i guess. Better is to block slowly, semidry, oldschool over several hours for storage. In any case you need to take care that the membranes stay as they are! like adding a stable plastic piece, your notebook or anything so that they dont bend or get moved...

After thawing, I strip the membranes heavily (2% (w/v) SDS; 62.5 mM Tris-HCl; 100 mM b-Mercaptoethanol or better DTT: 1g per 50 ml added fresh; pH 6.8 for 60-120 min. at 37 °C very mild shaking a bit), wash the membrane 2-3 times for 10 minutes per wash with TTBS and put them again in blocking buffer (i think this can be skipped maybe because blocked is blocked for nitrocellulose and will never ever change! but mhhh) as before. And i continue with normal staining...

I usually repeated that for three times (two freeze steps, with the most sensitive staining like anti-phospho before freezing) and one gets still unchanged publication quality (whatever that is nowadays...).

Sometimes I repeated it many (up to 8) times with some membranes. It depends on your epitope and your antibody, how sensitive it is to minor changes of the amino acid side chains, which happens over time. But with standard mabs like anti-CD3e, TCR, ZAP-70 it doesn't matter so much.

But I tested some old membranes (you know, the very old never ending projects) after up to 8 years in -80 and they still worked like new but with a really ecellent mab. I also have pics if anyone is interested...

I know some virologists reuse strips from gels extensivly over years with a certain virus seperated on it.

One serious question:

In most transfer buffers there is like 25-48 mM Tris and 39-192 mM glycine (Bjerrum and Schafer-Nielsen transfer buffer for SDS-proteins using nitrocellulose and Towbin transfer buffer respectively). I don't understand this, as Tris and glycine will react with nitro-groups and inactivate them. Why this is used?

There is also this Dunn carbonate transfer buffer based only on carbonate which should not react with the membrane. Did anyone try???

I use a modified version only (half Towbin):

12,5 mM Tris

96 mM glycine

10 % methanol

pH 8.3

keep it in refrigerator

use only TBS with azide (0,05 to 0,1%) in 4ºC, no more than 2 days. If you need more time, you can also freeze the membrane involved in plastic or with tbs.

Both PVDF and NC membranes can be air-dried after transfer, before the blocking step. They keep quite well even at room temperature for weeks or even months, if protected from dust (recall that house dust is mostly of human origin and would contaminate). I use polythene Ziploc bags. In the dry state, very little can happen to your proteins...

Once you have started the development (blocking, incubation with antibodies), continue until you have your bands. Then strip the membrane and air dry.

After drying, PVDF is usually wetted with methanol, because it is hydrophobic and will not accept water. However, you can use this to your advantage and probe the unwetted PVDF. The antibody can then interact only with the proteins on the membrane surface, not with the membrane itself. Therefore, no blocking is needed and the background is lower (doi:10.1006/abio.1999.4453)