The most standard phospho-antibody protocols involve blocking with 5% milk in PBS or TBS (0.1% Tween 20), followed by a couple washing steps with PBS or TBS to remove the excess milk. If you dillute your antibody in a milk solution, storage and re-use will be extremely problematic, because the milk will "go bad" and fall out of solution, similar to milk at home. BSA is ideal for your needs. Dillute your primary antibody in 5% BSA in PBS or TBA (0.1% Tween 20). If you add 0.05% Sodium Azide (Read the MSDS carefully), then you can store your dilluted antibody at 4C for years with no problem. The Azide will prevent any microorganism from growing. The one problem with Azide is that it inhibits the HRP enzyme, so definitely don't dillute your secondary antibodies in the same solution. Washing 3 times between primary and secondary antibodies is enough to get rid of the residual Azide. When I was a post-doc, we wrote up a good western protocol in various European languages with most of these concepts included: https://sites.google.com/site/pasteuralternativecareers/consulting-projects/the-european-western-blot-project   

Generally milk is not used for blocking if its a phospho antibody....

You can store used antibody in 4C for upto 2 weeks..

Use 1% non-fat milk for antibody preparation. If you want to use further then give pri Ab treatment at 4 deg for overnight or at least 4 h. Later, store at -20 deg. You can use easily 2-3 times. It works for most of the antibodies.

Best wishes

The most standard phospho-antibody protocols involve blocking with 5% milk in PBS or TBS (0.1% Tween 20), followed by a couple washing steps with PBS or TBS to remove the excess milk. If you dillute your antibody in a milk solution, storage and re-use will be extremely problematic, because the milk will "go bad" and fall out of solution, similar to milk at home. BSA is ideal for your needs. Dillute your primary antibody in 5% BSA in PBS or TBA (0.1% Tween 20). If you add 0.05% Sodium Azide (Read the MSDS carefully), then you can store your dilluted antibody at 4C for years with no problem. The Azide will prevent any microorganism from growing. The one problem with Azide is that it inhibits the HRP enzyme, so definitely don't dillute your secondary antibodies in the same solution. Washing 3 times between primary and secondary antibodies is enough to get rid of the residual Azide. When I was a post-doc, we wrote up a good western protocol in various European languages with most of these concepts included: https://sites.google.com/site/pasteuralternativecareers/consulting-projects/the-european-western-blot-project   

Dear Revathy!

I usually use (5% BSA in TBS-0.05Tween 20) for anti-phospho antibody. You can store it at  -20.

Both myself and my lab mates dilute our primary antibodies in 5% BSA in TBST with Sodium Azide stored at 4C for months to over a year and typically use them several times a month to weekly with great results.

Hii, Dilute primary antibodies in TBST with 5% BSA. Usage of milk can create non-specific background, because casein itself is a phophoprotein. Further, milk in TBST can not be stored for long at 4 degree C. you can store your primary antibody at 4 degree C for 2-3 weeks and can reuse 2-4 times efficiently.

As many of them mentioned here 5%BSA in TBST and you can store your diluted antibody at 4'C for 1 or 2 days.

Adding glycerol to your antibody 1:1 volume wise may help in avoiding adverse freezing and thawing effects after the first use.

We used to freeze our diluted antibody solution (in BSA- or milk-based blocking solutions containing azide) at -20°C if we didn't need them the next days again. Thaw again when you need the antibody, we used hand-warm waterbaths for thawing. How many freeze-thaw-cycles will work, depends on your antibody, but normally it will be several.

If you need the antibody dilution everyday, storage at 4°C will be better.

It all depends on how strong the signal you get with the previously used antibodies and how stable they are. Also, it depends on the working titer of the antibodies and hwo much of the antibodies were consumed during your first use of them. My advice to everybody is to standerdize that by rechecking the reactivity of previously used antibodies on strips from the same target protein and see if the signal remains as strong or gets weaker upon use.

Good luck

I agree with Mark. Using BSA and Sodium Azide should be an alternative.

I agree with both answers. I usually reuse my antiboyes. It dependes in how strong is the signal you get, in order to define how many times you can reuse it. At least two to four times for almost every antibody works for me. As for actin antibody even more. I always store them in TBS'0.1% Tween suplemented with azide for avoiding fungi growth.

To reuse the antibody, add 1:1000 of 10% Sodium Azide (Stock=10% Sodium azide in water).

Great answers here.  We design and produce phospho antibodies in our lab.  We block our antibodies with serine and threonine phospho sites with 5% non-fat milk.  We use BSA for phospho tyrosine antibodies because the phospho tyrosine in milk can produce high background.   It is true that milk goes bad faster than BSA, and BSA will certainly work for all antibodies.  The azide is great as long as it doesn't interfere with your system.  We divide our antibodies into working aliquots, and only keep one aliquot in 4 degrees at a time.  As mentioned in other posts, we add glycerol to 50% and store at -20 to avoid freeze/thaw.  For long term storage, we recommend aliquotting and storing at -80.  We generally don't reuse our antibodies, but here is an idea to reduce the amount of antibody you use when testing multiple antibodies using the same lysate: Try a gel with a stacking layer that has one large trough instead of one that has multiple lanes. Once the gel has been transferred to a blot, you can cut the blot into strips of equal widths. In most cases, twice as many strips can be cut from one large trough than from cutting apart a blot with multiple lanes. The strips are also more narrow and require much less incubation solution. 

In our experience with anti-phosphospecific antibodies the blocking with 2-5% BSA is the best. You might want to keep them at 4 °C with sodium azide as suggested, but you can also freeze them at -20 °C, the high protein concentration obtained with the BSA will protect them well enough from denaturation and degradation... but it should be tested on the specific antibody which way is better. Again in our experience, anti-phosphospecific antibodies cannot be re-used too many times, normally after 3-4 times they lose efficiency. But it could be related to the ones we use and their concentration... We prefer buffering in Tris, not in PBS, since we use pH 8.2, which strongly favours antibody-antigen interaction.

For all other antibodies I have to say blocking in 5% skimmed milk is far better than BSA, mostly with sera (BSA is not really blocking at all, but it's good to keep protein conc. high and protect the antibodies from degradation while storing), and freezing the used aliquot works in most cases really well. We regularly re-use the aliquots 4-10 times, depending on the antibody.

I have reused many different antibodies but have noticed the anti-phosphoprotein antibodies never work as well if not fresh

Yes you can reuse the antibody but again it depend on how much signal it pick . But how many times you can reuse that  need to be standardized. You can use minimum for two times.

yes yo can.but you have to store it with sodium azide.

During my MS after western blot i used to store the antibodies as it is @ 4C. But you can use it couple of times only, although sodium azide will increase its longevity by not letting the MO's grow. 

I completely agree with Mark. Nice answer and complete.

I would like only to suggest, that usually phospho antibodies work better in BSA solution than milk because you get less background.

Good luck!

For safety I'd only ever use them once more and within the same week.

I stored my antibodies in milk at -20°C and I used them at most 3 times, and within 2 weeks (if it's a good antibody). 

Thank you all for the valuable inputs

As other said, BSA is better for phospho antibodies and you could store them for a bit with azide

It depends on the antibodies. Some antibodies I can resue it for up to 5 times and within 2 months. Primary antibodies were applied after blocking in either 5% milk or 5%BSA and 2 washes in TBST. I don't add azide. To save money and research materials, 1ml of diluted antibody per membrane is good enough using pouches and sealer.

We also re-use our primary antibodies. All of them are in milk with sodium azide and we store them at -20°C. When in use, we keep them at 4°C. Depending on the antibody, even the phospho-antibodies work well multiple times. 

We have re-used antibody solutions multiple times (NaAzide) occasionally adding additional 1st antibody to solution since some dilution inevitably occurs. 

Like Amy suggested, you can save on the volume of each antibody by cutting strips using sharp scalpel and straightedge on a glass plate from blotted membrane from a "trough-loaded" gel. Mark top and bottom of gel/membrane with some methyl green-glycerol solution (experiment on timing as it will compete for protein binding to a membrane).  Bio-Rad at least used to sell disposable plastic trays with long narrow wells that can be reused for the binding steps. 

You can definitely re-use the primary antibody solution either by adding NaAzide to the solution and keeping it at 4°C or by freezing it at -20°C. Most antibodies can support up to 4-5 freeze-thaw cycle so it is best to keep track of this. However, you will see that some antibodies only work once.

i would suggest to use 3-5% of BSA  prepared in TBST For blocking if u are working with phospho proteins( Phosphorus present in milk may interfere with ur protein to gib false positive result). For storing ur antibody,add  Na- azide in a  final concentration of 0.02% in the antibody solution or in the blocking solution which u use to prepare ur antibody solution.i have used 8-10 times. But it may depend upon from where u r buying ur antibodies.Cell signalling are pretty good for this.Abcam and santa cruz are not as good as cell signaling if u want to re-use ur antibody

how much NaZide should be added to prevent your milk from going bad?

0.02% sodium azide as suggested before and keep it on 4C.

I think you can re-use your diluted antibody and stored at - 20. Also, for phospho-protein blotting, it is suggested to use BSA and dilute your primary antibody gently. According to our previous experience, we always use 5% milk to dilute the secondary antibody to reduce unwanted band on our membrane. Wish the best for your laboratory work.

I add Na azide (1:1000) to my 5% BSA in 1xTBST to prevent contamination while storing and reusing my primary antibodies.