PVDF vs. Nitrocellulose for Western blot - (Jan/31/2009 )

rkay447 on Feb 1 2009, 07:53 AM said:

Do you block after the wash steps right before you add the secondary? How long do you block for in the goat serum?

I've got a question about PVDF membranes. I've read a couple forums on the internet, but haven't found this in any descriptions or protocols for PVDF membranes. Following transfer, can you allow the membrane to dry, and then do the probing with your antibodies? I've read that you don't have to block if the membrane is dry. The reasoning is that once the membrane is dry, proteins will not bind to it (therefore, no antibody background) and the antibodies will only stick where the target antigen is on the membrane.

Has anyone done this or have any experience to say whether or not this would work?

Mr Jefferson on Jan 31 2009, 02:07 PM said:

Some ECL formulations don't like tween, so before adding the ECL do a wash on distiled water or PBS/TBS.
Never allow the membrane to dry during primary or secondary antibody incubations, the proteins would bind to the PVDF.
As you already did, reduce antibodies concentration.
You can try blocking with PVA.
Increasing blocking or tween reduces background, but may remove your protein from the membrane.

To improve detection limits don't use any protein blocking and reduce tween20 to 0,01% just block with PVA (130-180k) 1-100parts per million 1minute prior to primary antibody incubation. You can also microwave the membrane slightly in stripping solution to improve detection (before blocking).

I suggest activating in ethanol instead of methanol, it works equal or better and is enviromentaly friendly.
After this activation remove the ethanol by briefly shaking in water-buffer.

Letting the membrane dry after transfer may help to retain the proteins on te PVDF.
To reduce background perform short film exposure, to detect " the undetected" expose longer 2h-overnight.

Smack my face if I am wrong here: This is all ANECDOTAL EVIDENCE! Why is research littered with non-fact based opinions? There have been studies done by "electrophoresis/transfer studs" elucidating many of these matters (transfer buffer, wet vs. semidry, nitro vs. PVDF etc). Charged nylon appears to be great depending on the application. 0.2 um pore-size nitro has a higher binding efficiency than PVDF (presumably 0.45 um). Of course binding is dependent upon the individual protein itself, but in general there are some clear-cut info out there.

This is just one example and perhaps not the best (perform the PubMed search for other articles)

Electrophoresis. 1990 Jan;11(1):46-52.

Important parameters in semi-dry electrophoretic transfer.
Jacobson G, Kårsnäs P.

HERE IS SOME CONTRADICTING EVIDENCE (Remember that Pluskal has a patent on Immobilon + he washes the membranes after transfer 15 min at pH 10);

J Acquir Immune Defic Syndr. 1988;1(4):333-9.

Improved HIV antiglycoprotein antibody detection by immunoblotting on a hydrophobic membrane.
Lauritzen E, Pluskal M.



As a rule I would suggest a two-step wet transfer with 0.2 um nitro membranes as described by (the question is do you have the patience?):

Anal Biochem. 1987 May 1;162(2):370-7.

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins.
Otter T, King SM, Witman GB.

Everybody: Have a nice day and quit the anecdotal evidence thing. You are confusing new and upcoming students/researchers including myself.

smack...

what you are calling anecdotal we are calling experience. don't let the "studs" fool you, as you pointed out, some of them have vested interests in the results they report.

you can read all the papers you want (and we have) but nothing beats experience.

and, by the way, as you continue your career in science, you will find that many authors omit parts of procedures that may be important (even critical), intentionally or not.

so, when someone gives you the benefit of their experience it may be wise to, at least, take it into consideration rather than just write it off as "anecdotal".

mdfenko on Feb 12 2010, 11:26 AM said:

I did not mean to offend anyone (anecdotal was kind of harsh I agree and perhaps not very truthful in the case of hard-working students/techs/ scientists). You did miss the message by shooting the deliverer I think and it is my fault. Anyhow, you are partly correct (commercial interests, purposely omitting procedures ), but many laymen opinions when it comes to WB are indeed not evidence-based. The general problem (it seems to me at least) is that when people get their first successful blot (after many hours of painstaking beginner's mistakes) they stick to the same procedure for the rest of their research career. What is worse is that they spread their "Western Blotting word" like it is the gospel, quran, the God delusion or whatever rocks their boat. In general, we are not doing the students any favours by blindly advocating our often ill-researched WB protocols .

In one really long sentence: Most students/and beginning researchers really have little to no clue what electrophoresis/transfer conditions yield the highest amount of low and high MW proteins in gels/membranes (gradient gel vs. single % gel?, equilibrate gel in transfer buffer or not, semidry vs. wet transfer? how long transfer? what transfer buffer? nitro vs. PVDF vs. charged nylon? what pore-size binds the most proteins if you only can pick one?), what blocking solution to use (milk, BSA, casein, detergent? 1,2,3,4,5% for 1 hour? 10% milk for 10-15 min?), why or why not detergent should be used in wash steps (does it interfere with ECL?, 3 x 5 min? 3 x 20 min? 5 x 20 min?, in which wash-step do you use detergent?, Tween20? Triton X100), how to trouble-shoot amount of Ab and ECL (can you really expect signal to show up when waiting 10 min to develop ECL'd membrane previously incubated with 1:2000 secondary Ab), which developing system to use (traditional film or "gel-box"?, which one is really more sensitive?), how to strip the membrane (mild or harsh stripping buffer, is the commercial RESTORE harsh or mild?), and how to store the membrane (wet or dry and for how long?).

There are so many opinions out there and the reality is that only a few people have actually done the studies or the tedious troubleshooting . Until people publish their observations then I would almost call it hearsay i.e. anecdotal evidence. Well all know this: The least one could do as an aspiring scientist is to read these pubs and then formulate our own protocols by taking the best parts from each pub. Thereafter we can trouble-shoot our "optimized" WB protocols and compare to what is traditionally presented by the general public.

Regarding commercial interests: Take the Otter paper in my previous list.....I'm not saying it is any spectacular, but it does show that high and low MW proteins can be captured on one and the same membrane . Several experts are still saying that this is impossible unless you use the Invitrogen system. I don't think these guys (Otter) had any commercial interests. At the very least they published their findings in a peer-reviewed paper for the benefit of those who want to base their research on published findings rather than what I (in)accurately referred to as 'anecdotal evidence'.

Take care!

-Mats_Nilsson-

Mats_Nilsson on Feb 19 2010, 02:50 PM said:

mdfenko on Feb 12 2010, 11:26 AM said:

smack...

what you are calling anecdotal we are calling experience. don't let the "studs" fool you, as you pointed out, some of them have vested interests in the results they report.

you can read all the papers you want (and we have) but nothing beats experience.

and, by the way, as you continue your career in science, you will find that many authors omit parts of procedures that may be important (even critical), intentionally or not.

so, when someone gives you the benefit of their experience it may be wise to, at least, take it into consideration rather than just write it off as "anecdotal".

I did not mean to offend anyone (anecdotal was kind of harsh I agree and perhaps not very truthful in the case of hard-working students/techs/ scientists). You did miss the message by shooting the deliverer I think and it is my fault. Anyhow, you are partly correct (commercial interests, purposely omitting procedures ), but many laymen opinions when it comes to WB are indeed not evidence-based. The general problem (it seems to me at least) is that when people get their first successful blot (after many hours of painstaking beginner's mistakes) they stick to the same procedure for the rest of their research career. What is worse is that they spread their "Western Blotting word" like it is the gospel, quran, the God delusion or whatever rocks their boat. In general, we are not doing the students any favours by blindly advocating our often ill-researched WB protocols .

In one really long sentence: Most students/and beginning researchers really have little to no clue what electrophoresis/transfer conditions yield the highest amount of low and high MW proteins in gels/membranes (gradient gel vs. single % gel?, equilibrate gel in transfer buffer or not, semidry vs. wet transfer? how long transfer? what transfer buffer? nitro vs. PVDF vs. charged nylon? what pore-size binds the most proteins if you only can pick one?), what blocking solution to use (milk, BSA, casein, detergent? 1,2,3,4,5% for 1 hour? 10% milk for 10-15 min?), why or why not detergent should be used in wash steps (does it interfere with ECL?, 3 x 5 min? 3 x 20 min? 5 x 20 min?, in which wash-step do you use detergent?, Tween20? Triton X100), how to trouble-shoot amount of Ab and ECL (can you really expect signal to show up when waiting 10 min to develop ECL'd membrane previously incubated with 1:2000 secondary Ab), which developing system to use (traditional film or "gel-box"?, which one is really more sensitive?), how to strip the membrane (mild or harsh stripping buffer, is the commercial RESTORE harsh or mild?), and how to store the membrane (wet or dry and for how long?).

There are so many opinions out there and the reality is that only a few people have actually done the studies or the tedious troubleshooting . Until people publish their observations then I would almost call it hearsay i.e. anecdotal evidence. Well all know this: The least one could do as an aspiring scientist is to read these pubs and then formulate our own protocols by taking the best parts from each pub. Thereafter we can trouble-shoot our "optimized" WB protocols and compare to what is traditionally presented by the general public.

Regarding commercial interests: Take the Otter paper in my previous list.....I'm not saying it is any spectacular, but it does show that high and low MW proteins can be captured on one and the same membrane . Several experts are still saying that this is impossible unless you use the Invitrogen system. I don't think these guys (Otter) had any commercial interests. At the very least they published their findings in a peer-reviewed paper for the benefit of those who want to base their research on published findings rather than what I (in)accurately referred to as 'anecdotal evidence'.

Take care!

well, then. i guess that maybe some should pay attention to those of us who have done the research (published or not).

by the way, there are also several books, pamphlets, pdfs,... which synopsize the various issues to take into consideration and contain troubleshooting guides so that we don't all have to reinvent the wheel.

-mdfenko-

You habitually require to hydrate PVDF with methanol and rinse with water (deionized) until it doesn't bead before transfer. Otherwise proteins don't attach rather as well. This might be part of your problem. What does the ponceau gaze like after transfer

-shane-

yobou on Mar 14 2009, 11:21 AM said:

I have been using PVDF membranes for more than 3 years now, and its very fine. after I develop the membrane by ECL reagent or millipore regant and filming it, I store it at refregerator (for days) until I strip it for the next antibody. For blocking you can use either 3% skim milk for 1 hr at RT, or 10% skim milk for 10 minute at RT.

just another question regarding PVDF
does the membrane take long to stain in ponceau.
and whenever i do a ponceau stain of my PVDF the bands appear like a shawdowy black colour instead of the usual pinkish-red colour of the ponceau.

-eurisko-

little late on this one. yes, we're all capable of sifting through protocols and publications in an effort to trouble-shoot but nothing compares to knowledge gained from years of experience. furthermore, i thought the whole point of this forum was to take advantage of other people's experience and to save time?

1. rinsing off the MeOH pre-wet from PVDF seems pointless to me since you usually soak the membrane in transfer buffer just before sandwiching and transfer buffers typically contain 20% MeOH

2. SDS inhibits protein transfer to nitrocellulose membranes. Can aid transfer of large proteins to PVDF

-WblotMaster-

Pages: Previous 1 2 3 Next