Talk:Western blot

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This article was the subject of a Wiki Education Foundation-supported course assignment, between 2 February 2021 and 14 May 2021. Further details are available on the course page. Student editor(s): Farooqak10. Peer reviewers: Farooqak10.

Above undated message substituted from Template:Dashboard.wikiedu.org assignment by PrimeBOT (talk) 12:52, 17 January 2022 (UTC)

Old stuff
Westerns are not only used in molecular biology ;-), perhaps "protein chemistry" may be more appropriate (from Hennef, Germany)

How is the word incubate being used here? What keeps the protein of interest from washing away? Why does binding with an antibody make the protein stick to the nitrocellulose membrane?

Western should be written like western, not Western because it does not come from a name like Southern blotting.
 * The commericial instructions sitting on my desk say Western is correct. Ladlergo 20:16, 4 April 2006 (UTC)

proteins sticking to the membrane
The article says:

The membrane is "sticky" and binds proteins non-specifically

You may have previously seen proteins stuck to surfaces.....maybe blood stains or grass stains. The plastic membranes that are used for a Western blot hold onto the proteins tightly and you do not have to worry about the proteins washing off of the membrane during the antibody steps.

The antibodies have nothing to do with the target protein(s) staying attached to the membrane. The antibodies specifically bind to their target protein by a molecular lock-and-key type of interaction. The antibodies that are used for Western blots typically have a slow rate at which they detatch from their target protein, allowing them to stay attached through the various incubation and rinse steps.

also known as an immunoblot

I noticed that "Tissue preparation" is listed as the first step the immunoblot (western blot). It should be "Sample preparation". I've done hundreds of immunoblots, but only a few from tissue culture cells. As a molecular microbiologist, most of my samples are from yeasts, subcellular fractions of yeasts, or their culture supernatants. There are a lot of other samples besides just 'tissue' —Preceding unsigned comment added by Yeastpics (talk • contribs) 11:08, 30 August 2010 (UTC)

er...
For those confused by my edit summary it should read "that I think's most common", but some fool decided to put the apostrophe key next to the enter key! Joe D (t) 16:57, 10 January 2006 (UTC)

Alan Au Editing
User Alan Au, seems to hate Molecular Station no matter if the pages have quite a lot of information. I believe Alan Au has no idea about bioinformatics or any other molecular biology information. He believes RNA Bioinformatics is not "accepted by the scientific community" and removed my contributions to that page. See Bioinformatics Talk.

Compare these pages: Western Blot

Western Blotting. They both have a lot of information, although one was deleted. See discussion at Alan Au's Editing.

Wikipedia will never be a good resource for scientists, researchers and the general public as we do not have highly educated (Ph.D's, M.Sc's) individuals contributing as many of their contributions are being deleted.

I am a PdD who ran western blots a year or two after they were first described, and i have contributed to this article..I don't have the energy to compeltly re write it (needed) esp since my technically correct rewrites get trashed by people who write things that are just plain wrong. Having said all that, as sicentist who does biotechnology, I, and to my perosnal knowledge, other scientists, often rely on wkipedia as a first line of info.Cinnamon colbert (talk) 22:06, 22 February 2009 (UTC) (excuse the poor spelling of this note)

Bioinformin

Chemiluminescence != fluorescence
Just a quick nitpick... chemiluminescence and fluorecence are not the same thing, although they do have similar photophysical mechanisms. Fluorescence refers to the excitation of a molecule to an excited state, and the subsequent decay by radiative emission from that excited state. Chemiluminescence refers to the situation where a molecule is formed in an excited electronic state as the product of a chemical reaction, and subsequently decays by radiative emission to the ground state. There are a couple of instances in the article where chemiluminescence and fluorescence are mentioned as though they are interchangeable... not a big deal, just thought I'd mention it. -Oli 203.63.0.143 23:36, 11 June 2006 (UTC)
 * or, leaving aside the technical mumbo jumbo...in fluorescence, a molecule absorbs a particle of light (a photon) and then emits a second photon. The energy of the 1st photon is greater then the energy of the 2nd photon.

In chemiluminescence, a chemical change releases energy that is "stored" in a molecule, and the released energy appears as a photon. This is highly unusual, as most chemical changes that release energy (eg, burning of gasoline) result in the release of heat. —Preceding unsigned comment added by Cinnamon colbert (talk • contribs) 13:34, 25 February 2009 (UTC)

Consistency?
From the Southern blot article: "As the technique was eponymously named, Southern blot should be capitalised, whereas northern and western blots should not."

--Gycklaren 10:48, 27 May 2007 (UTC)

The CDC page on writing style says that capitalized Western blot is the preferred. I'm sure one can make arguments for or against capitalizing, but usually style should conform to one standard and the CDC is a good enough authority. Calgirl2 (talk) 18:20, 29 November 2007 (UTC)


 * It was capital W when I was a student in the 80s. 2A01:CB0C:CD:D800:60E4:E8AC:CCF6:6E79 (talk) 14:34, 19 January 2021 (UTC)

Article picture
The picture on the right seems to be missing? --David Munch 13:20, 17 June 2007 (UTC) 152.131.10.197 (talk) 02:06, 23 August 2012 (UTC)

Are you sure the picture is of a Western and not a Southern? It looks like a picture taken with Ethidium bromide under UV light, and the ladder appears to be too small. Westerns usually run from 200 to 30 kDa or so. I could be wrong, it just doesn't look right to me. Please correct me if I am mistaken.

152.131.10.197 (talk) 02:06, 23 August 2012 (UTC)

Protocol section
Please contribute links to the protocol section. Pages that describe common molecular/biochemical techniques will benifit if we make an effort to add links to the best protocols. What do other people think?

Capitalization
Should it be "western blot" or "Western blot"? Strictly speaking "western blot" does not seem to be a proper noun. However, the CDC journal of style (http://www.cdc.gov/ncidod/EID/style_guide_p2.htm) supports using a capital W in "western." I've seen it written both ways in both scientific articles and prose. Science, the premiere American science magazine, uses a capital W and Nature, the premiere British one, uses lowercase. Thoughts?12.144.50.194 (talk) 17:55, 21 February 2008 (UTC)
 * I checked a the style guides for submitting to various science journals and the CDC website and most capitalize Western blot. The World Health Organization, however, does not capitalize eastern, northern or western blot, only Southern blot (named after the biologist). This article uses a mixture of cases so I changed them all to upper case - feel free to change to the lower case as long as they are consistent. Dr d12 (talk) 20:07, 9 May 2008 (UTC)

My understanding is that Southern blots were named for EM Southern, hence the capitalization, whereas technically similar RNA and protein hybridization protocols were named northern and western blots, respectively, as cutesy take-offs. —Preceding unsigned comment added by 144.74.213.203 (talk) 16:00, 5 January 2009 (UTC) Neal Burnette invented the term and spelled it 'Western blot' (capitalized). If anyone knows how it's spelled, it's probably him... 129.67.74.57 (talk) 18:27, 11 October 2011 (UTC)
 * I'd say that if Burnette spelled it that way we should go with it, because that's probably the most definitive answer we will get. However, didn't he capitalise the entire term, i.e. Western Blot instead of Western blot? Unfortunately I have no access to the paper ATM, but maybe someone can check this: . -- Shinryuu (talk) 21:38, 11 October 2011 (UTC)
 * In the paper 'Blot' is only capitalized in the title (along with 'Proteins', etc., as is usual for titles). In the remainder of the article it's 'blot' and 'blotting'. 129.67.74.57 (talk) 10:53, 12 October 2011 (UTC)

Having read the original paper, and getting pissed off with my supervisors for correcting me (I wrote it with lower-case), I can confirm that it is indeed upper case, since this is how the original author/inventor wrote it throughout the paper. — Preceding unsigned comment added by 124.182.92.253 (talk) 00:26, 1 May 2013 (UTC)

Western blot origins
I have been looking for references for this affirmation:


 * The method originated from the laboratory of George Stark at Stanford.

However, the first reference I find for first Western blot is: Towbin H, Staehelin T, Gordon J. (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications." and can't find any George Stark's article talking about anything similar to Western blot.

Shall anyone explain this?

PS. Sorry for my poor English.--EKhan (talk) 11:41, 17 August 2010 (UTC)


 * In the abstract to the paper by Stark et al. cited here on the Western Blot Wikipedia article, it says:
 * "We describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently." (Article at PMC)
 * Except for the transfer to the membrane being different today, this sounds exactly like Western blotting to me.
 * -- Shinryuu (talk) 12:08, 17 August 2010 (UTC)


 * Yep, I didn't noticed that one, I should have searched (much) better. Thank you a lot! --EKhan (talk) 13:43, 17 August 2010 (UTC)

A review on western blotting from 2006 describes Towbin as the author for W.blotting and Stark for Northern
 * -- maxhebditch 14:10, 07/01/13 —Preceding undated comment added 14:12, 7 January 2013 (UTC)

SDS-PAGE and Transfer Diagrams: wire colors
The anode and cathode are labeled incorrectly in the picture. Anodes are negatively charged while cathodes are positive. — Preceding unsigned comment added by 99.10.123.53 (talk) 15:19, 1 December 2011 (UTC)

In response to above comment: the anodes and cathodes are labeled correctly. A cathode attracts cations, which means it must be negatively charged. An anode attracts anions, which means it must be positively charged. This is counterintuitive but completely correct.

On an unrelated note, the picture describing the section on Transfer (currently section 1.3) has an error. The order of the gel and nitrocellulose membrane should be flipped. Since proteins become negatively charged when coated by SDS, the proteins will travel towards the cathode (away from the membrane, in this diagram). — Preceding unsigned comment added by 131.215.3.182 (talk) 20:28, 31 July 2013 (UTC)

Response to both above parties: Labelling and writing on anodes and cathodes look more or less correct (I have revised relevant sections a little to eliminate ambiguity). The issue causing confusion with both the SDS-PAGE and Transfer diagrams is the choice of colors for the wires (maybe same could be said for color of buffers in SDS-PAGE fig). I believe we can come to consensus that SDS shell around proteins will be negatively charged (anion) and that anions migrate towards the anode, by definition. However, the pole that anions migrate towards is by convention called positive (opposites attract rule) and is red (maybe there is an IEEE or other guideline for this, not sure), with the other pole (cathode) being black, blue or brown. For both SDS-PAGE and transfer blotting equipment, proteins migrate towards the red electrode on all equipment I have seen. It is possible to connect some of the Bio-Rad equipment in the orientation these Wikipedia diagrams show. If a student does that (and it has happened), the power supply will still function as if all is well, but experiment will fail. Students will insist they did everything correctly because they did it just like the Wikipedia article. It also leads to students being confused about how the underlying chemistry works. Electronics engineers may not use the terms anode or cathode at all, in preference of assigning a red wire to the electrode with higher voltage. I would like to see the colors reversed on these diagrams to prevent future confusion. If I am somehow in error, please accept my apology in advance. Sincerely, Dominic-Luc Webb — Preceding unsigned comment added by 79.102.170.83 (talk) 06:32, 22 June 2018 (UTC)
 * Regarding use of colors, your comment lines up entirely correctly with my own experience (as well as a corresponding Google image search for "electrophoresis power supply"). Positive (+) leads and sockets are virtually always red; negative (-) leads and sockets are nearly always black or (occasionally) blue.
 * Unfortunately, the creator of File:SDS-PAGE Electrophoresis.png and the other images hasn't edited Wikipedia for about five years (and the images themselves were uploaded almost ten years ago) so it may be difficult to readily modify the images. Someone may have to redraw them entirely.  (Truth be told, we could probably do without the hamilton syringe sample loading, too&mdash;does anyone still do it that way?) TenOfAllTrades(talk) 02:53, 26 June 2018 (UTC)

getting worse with time
as a PhD who was one of the first people in the world to actually do a western with my own two hands, shortly after Towbin et al, this article is getting worse with time; it used to be better !!!!! (and I do know what i am talking about; I know that Ponceau S is now the preferred post blot protein stain, vs the commercial product from pierce (anthranavally et al) or direct blue/black or eosin or copper based stains; I know that BCIP/NBT fades on a dry pvdf blot, but that DAB is supposed to be toxic, ...) the fancy graphics are just bad I know, it is wiki, don't complain, fix it, but I get tired of having to re re re re correct stupid errors put in by others. — Preceding unsigned comment added by 50.195.10.169 (talk) 18:03, 21 March 2013 (UTC)

you can not be getting tired of correcting errors because you never have. Check your edit history. explain why the graphics are bad. Bensaccount (talk) 23:53, 26 March 2013 (UTC)


 * I seriously doubt you're an expert on the subject. I think you're a troll who has nothing better to do than complain about wikipedia articles. If you are indeed an expert, make an account and contribute, instead of complaining. Higgyrun3 (talk) 00:03, 27 March 2013 (UTC)

Yo folks, these graphics are like mega bad and way out of date. 129.109.65.148 (talk) 03:18, 10 September 2013 (UTC)Anon-but like I run thirty of these a month and it is baaaaaaaaad.

I changed a lot of the paragraphs to reflect modern methods of western blotting. Please add relevant citations at your leisure. — Preceding unsigned comment added by 129.109.65.148 (talk) 03:45, 10 September 2013 (UTC)

RIBA
Does the term RIBA have anything to do with this topic?

I have edited the Royal Institute of British Architects article to change:

to

But the Riba (disambiguation) page does not list this page.

If a user is likely to search for this topic using "RIBA", then please add this article to Riba (disambiguation). Verbcatcher (talk) 01:54, 16 February 2014 (UTC)

Nature Methods paper to consider for inclusion
--User:Ceyockey ( talk to me ) 13:43, 14 September 2014 (UTC)

Contradiction
It seems that the article claims fluorescence is more sensitive than chemiluminescence but then claims the opposite in a later paragraph. Not sure which is correct, but it's something to look out for. — Preceding unsigned comment added by 143.215.227.205 (talk) 18:22, 31 December 2014 (UTC)

External links modified
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Automated Western blot technology introduction and overview
Hello All,

Would it be possible for me to introduce a technology that automates Western blots (http://www.proteinsimple.com/simple_western_overview.html)? It's been widely accepted in publications, journals, academia and industry folks. I want to show how Westerns can be performed via capillary electrophoresis.

Thank You, Steven — Preceding unsigned comment added by Steven.le57 (talk • contribs) 17:10, 9 May 2019 (UTC)

Controversy?
I'm no expert, but this looks relevant to include?

| Chinese ministry investigates duplications in papers by university president

|The Prevalence of Inappropriate Image Duplication in Biomedical Research Publications

— Preceding unsigned comment added by ChrisLambertUK (talk • contribs) 22:31, 27 November 2019 (UTC)

Wiki Education- Course Assignment
I am a Molecular Biology master student and, this is the article that assigned to me under the Recent Developments in Biotechnology Course. Further details are available on the course page.

Student editor: Sumydrmz

Sumydrmz (talk) 18:43, 11 December 2022 (UTC)


 * The course page link is: outreachdashboard.wmflabs.org/courses/Uskudar_University,_Istanbul,_Turkiye/MLC501_Recent_Developments_in_Biotechnology_2022-2023_Fall_(2022-2023)/home Sumydrmz (talk) 08:12, 20 December 2022 (UTC)