20 replies to this topic

[Mats_Nilsson]

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 [such is life]), but many laymen opinions [including myself] 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 [I am sure that yours is well-researched...that is not my point].

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[s] 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 [it takes too long time for the lazy students/researchers: 'let's just do what we did before']. 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 [which most people don't] 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 [just so happens using nitro 0.2 um]. 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!

Edited by Mats_Nilsson, 19 February 2010 - 12:31 PM.

[mdfenko]

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 [such is life]), but many laymen opinions [including myself] 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 [I am sure that yours is well-researched...that is not my point].

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[s] 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 [it takes too long time for the lazy students/researchers: 'let's just do what we did before']. 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 [which most people don't] 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 [just so happens using nitro 0.2 um]. 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.

[shane]

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

[eurisko]

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.

[WblotMaster]

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

Edited by WblotMaster, 03 June 2010 - 03:42 PM.

[mdfenko]

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

sds will aid in the transfer of large proteins onto nitrocellulose, too. it will also interfere with binding to pvdf (one effect is that it makes the protein travel too fast to be captured). methanol in the buffer strips it off the protein so that it can bind to either membrane.

you can get this and more information from the millipore protein blotting handbook.