Hi All,
I do store the membrane in a box filled with PBST, but the proteins on the membrane seems disappeared through time (after a few week or a month).
Is there a better way to store the membrane? for later stripped or exposed.
Thank you,
To store protein-transfered NC or PVDF membrane for a longer time, it is better to dry the membrane and then keep at -20oC without folding.
For PVDF membrane, just rinse in methanol before you blot. For NC membrane, put into TBST directly to blot.
To store protein-transfered NC or PVDF membrane for a longer time, it is better to dry the membrane and then keep at -20oC without folding.
For PVDF membrane, just rinse in methanol before you blot. For NC membrane, put into TBST directly to blot.
Dry the membrane and store it wrapped in aluminium foil or zip lock without folding in -20C
I think Xianfeng has answered the question. But even during the first week, try not to keep the blot in TBST. Keep it in 1X TBS at 4C instead.
For longer storage, wrap the dried membrane in aluminium foil and keep it at -20 C. If you want to blot it again within a week, you can also keep it at 4 degrees.
I have successfully re-probed PVDF membranes that were dried and kept at RT for years. So I wouldn't even bother with freezing it.
I have stored wrapped in glad wrap and a zip lock bag for over 12 months at 4 C, and re-probed successfully.
I used to wrapped in glad wrap and stored at 4C. So far it works for re-probed.
In some cases, the adding of some glycerol (1 - 3%) could be helpful. The last washing buffer should be free of detergents. The will go otherwise slowly between the protein and the solid phase (NC or PVDF) as you can observe also in ELISA.
Store the membrane dry but frozen between two sheets of filter paper. The presence of to much humidity will have two effects: 1. the same as the detergent mentioned above and 2. proteases can be still active. So will lose antigen at the solid phase.
If you store the membrane in a zip-lock you need for long time storage you should add a desiccant.
Like Tom Bender, after drying PVDF membranes I successfully reprobe them after they've been stored at room temperature for months. That way they are ready for stripping. To re-expose without stripping I would reincubate with the primary and secondary antibodies.
Wash them with 1% TBS after exposure. Allow them to dry. Put them in an envelope and stack them on the bookshelf along with the other paperwork. They work just fine for the next year or two.
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
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