Western Blotting

Подписаться 72 тыс.

Просмотров 1,1 млн

50% 1

For more information, visit www.bio-rad.com/yt/western-blo....
This video demonstrates SDS-PAGE separation of proteins using the Bio-Rad Comparative Proteomics Kit II: Western Blot Module. Assembly of the blotting sandwich and electroblotting are shown along with the steps for protein detection using a colorimetric assay.
www.bio-rad.com/evportal/desti...
We Are Bio-Rad Explorer.
Our Mission: Bio-Rad’s Explorer program provides easy access to engaging hands-on science learning experiences that spark interest in science and its influence on the world.
To do this we:
Provide high quality, relevant, learning experiences based on real world science
Empower educators with skills and confidence to deliver engaging, memorable lessons
Connect with Bio-Rad Explorer Online:
Website: www.bio-rad.com/en-us/education
Twitter: / bioradeducation
Pinterest: / bioradexplorer
LinkedIn: / 1613226
Facebook: / biorad
Instagram: @BioRadLabs
Snapchat: @BioRadLabs

Наука

Опубликовано:

15 окт 2012

Ссылка:

Скачать:

Готовим ссылку...

Добавить в:

Мой плейлист

Посмотреть позже

Комментарии : 131

@nikkibryceroque3026 6 лет назад

I only watched this because of my assignment and gosh I've never been so overwhelmed by such laborious procedures!

@medielijah 4 года назад

lab is not your metier, try something else, no shame

@resistance110 3 года назад

@@medielijah What a stupid comment you made. Discouraging someone because they can't do a western on their first try. Stay out of labs with that negativity.

@oscarqr1 3 года назад

@@resistance110 Honestly lmao. Almost everyone messes up protocols on their first try. Hell, I still mess up protocols after a few years of lab experience. Sometimes it's funny. Sometimes I destroy weeks of work and it's not so funny. Shit happens o.0

@yeny7194 3 года назад

@resistance110 and @oscar you guys are awesome

@ronaldokevin5693 3 года назад

@@medielijah social media is not good for you, try something else, no shame

@yodaydyxz1752 9 лет назад

Very informative Thank you so much for taking time and explain each step.

@michelebos5231 7 лет назад

Thank you for this video , it really helpes me to understand correctly what i'm doing, when and how. Last week was the first time i had to do this proces but it was all unclear to me. Now I know what to do . Thank you so much !

@beautifulmeena2571 5 лет назад

thnku sir

@lincolnoliveira3041 3 года назад

Yuka

@Hoxgene 2 года назад

That final result was beautiful!

@muhammadyudadwitamaagustia9883 4 года назад

when he was discarded the wash buffer or antibody, it gave me anxiety

@rowang5578 Месяц назад

He uhh he give me butterfly 🤭 those green hand

@starrynight7891 9 лет назад

Thank You so much for your wonderful video!

@hamlilasma5434 Год назад

all respect for this valuable work , thank you 🎩🌸

@EDUARDO12348 8 лет назад

Thank you BioRad

@noemimirra7907 7 лет назад

really enjoyed watching this video, great review!!! Thank you!!!

@user-rf4vc7mt4d 5 лет назад

WATCH IN X1.25 SPEED just to save you some time

@user-rf4vc7mt4d 3 года назад

Scam

@davidwaziri8460 2 года назад

Vedio

@mohammedal-hammadi5085 3 года назад

Thank you so much, it's pretty helpful

@arshisstudio1394 7 лет назад

thank u so much.. this video was so helpful

@adronung1892 4 года назад

What is the composition of blotting buffer, blocking buffer, wash buffer?

@joyjustman3536 Год назад

Wow! This is so helpful I must say

@marcussmith9461 5 лет назад

I have a question that might be answered here. I'm trying to get by cheaply and was wondering if I could run a western blot gel in a DGGE electrophoresis chamber ?

@jaimes6152 7 лет назад

THANK YOU.

@lmacunac 8 лет назад

Nice video about it!

@nurshahilinamira4165 5 лет назад

Nice video!

@p91grwm14 3 года назад

Do you use nitril gloves? Are they resistant in contact with methanol in the Buffer?

@lamhirhcn 9 лет назад

Add 10 mL of primary antibody and pour off it...reminding me of the time when I sealed 2 mL of the primary antibody along with the membrane for binding and then recycled the used primary antibody...

@MrSmokingHott 5 лет назад

Lamhirh amen to that!

@TheTillieTube 5 лет назад

This is how we do it in real life....

@ShakespeareCafe 5 лет назад

BioRad has a big budget and can afford to pour off the Expen$ive primary antibody...you can reuse it a few times before sending it down the drain

@seetheworld6656 Год назад

How to recyle it??

@NewWesternFront 11 месяцев назад

@@seetheworld6656 put it in the blue can on your house curb

@tutoradvance 9 лет назад

I see this for sale on the wards scientific , but says the kit does not include fish samples, I can't find where I am suppose to buy the fish samples from?

@SaSaRush 2 года назад

Very helpful thank you

@Dilmi24 9 лет назад

Thanks so much! :))

@Birs_84 3 года назад

Thanks for this video. It's been a while since I did my last WB, so thanks for a short refreshing. But don't you think the 10 mL of substrate solution were significantly more than the 10 mL of both antibody solutions? ^_^

@menglinzhao2536 2 года назад

Hi, I want to know what kinds of Substrate you used? The blue color you can see through the naked eyes or have you used a tool to analyze?

@govindmeena94140 4 года назад

Thanks Bio-Rad ....

@emilie6466 4 года назад

Govind Meena thanks Bio-dad...

@hanumakumar8997 2 года назад

Thank you

@ruths6022 3 года назад

Thank you sir

@dawoodsalar7202 5 лет назад

why we add scondry antibodies????

@jie0330 6 лет назад

it is 2018 and something has been updated ( instructions and so on)

@akansha4575 Год назад

Nicely explained 👏💐thnku

@ayeshasultana6391 7 лет назад

Thanks !

@user-fn7hs5yc2h 7 лет назад

Helpful video, thank you. A piece of question, though; I expected chemiluminescent detection or use of detection equipment in general to visualize the bands after the secondary antibody but the video shows direct color development through substrate addition, would you clarify?

@user-fn7hs5yc2h 7 лет назад

***** Thank you for the reply, point clarified.

@diogenesofsinope2692 Год назад

@@user-fn7hs5yc2h explain pls

@destroya3303 8 лет назад

a rocking board is that really necessary?

@laurentiaadinda8439 4 года назад

"pour off the primary and secondary antibody" me watching this: o m g, i sealed and reused for many times bcs expensive😂😭

@monishajayabalan7823 4 года назад

You are not alone 😂😂

@user-we9qg3dy5n 3 года назад

Same

@jatnarivas8741 3 года назад

Have you found the results to be equally reliable either way?

@HercadosP 3 года назад

@@jatnarivas8741 not as reliable. Have had some complete failures, but good enough for repeats to verify results. When I am ready to repeat the experiment for publication I use new antibodies

@ishaqadjaimi9645 5 лет назад

Thanks

@trilochans5008 2 года назад

How much duration of time take to diagnose western blot test for hiv in lab???(not about window period)

@1904Aparna Год назад

10ml of primary Ab and 10ml of substrate volumes were SO different, this does not feel right. I've never done a western blot, can someone explain which one was wrong? It kind of looks like 100ml of substrate

@ScienceGeek23 2 года назад

Cool gloves!

@stephenchetwynd7760 4 года назад

Im trying to buy one of the plastic cases that you build the transfer casette shown in the above video but cant seem to find anyone selling.. what are they actually called?

@BioRadEducation 4 года назад

Hi, Stephen! Please see www.bio-rad.com/en-us/product/mini-trans-blot-cell#fragment-4. If we can help further, call us at 1-800-4BIORAD and we'll help you find what you're looking for.

@yifanhu1546 8 лет назад

could anyone tell me what are the components of the substrate he added in the final step? it can direct see the band without exposure on machine. because for me ,after adding second antibody and washing, i incubate the membrane with immunostar or Chemi lumi One L, and then exposure.

@yifanhu1546 8 лет назад

+BioRadLifeScience i see, thank you so much!

@tony232cool 4 года назад

how do yo do detection after electrotransfer.

@twinkle1548 2 года назад

Thank u soooooooooooo much🙏

@DeepsikaP 2 года назад

Grt video! Can the antibodies be reused?

@puspanjali2464 Год назад

Yes you can

@pinkyflowe5762 7 лет назад

it helpful video. thnx

@JohnSmith-td7hd 6 лет назад

Reminds me of developing film.

@-..l 4 года назад

John Smith I agree, like developing film. Expect, it is not in a dark room.

@JM.5387 3 года назад

In the old days, we used photographic film, and developed it in a dark room. Everything was labeled with radioactivity.

@geetarani2875 5 лет назад

good

@user-bt8ru7rm9w 5 лет назад

can i use salmon sperm DNA for blocking solution?

@ConCerN.exe. 19 дней назад

👍 Helpful

@kasraakbari229 5 лет назад

wow!!!!! great

@xenawu1588 8 лет назад

what is the purpose of the blocking solution?

@nicheng1927 8 лет назад

+S Gill preventing nonspecific binding of primary and secondary antibodies in downstream steps. Blocking agents work by covering the unoccupied areas of the membrane with a dense layer of molecules. Blocking agents can either contain proteins, or be protein-free. I use to fatty-free milk(the cheapest and common one) or chick serum to be blocking buffer. If you want to improving your sensitization in phos-antibody, there have various commercial blocking buffer for that.

@pecatum666 10 лет назад

Why haven't you added any blocking solution?

@roseb2105 8 лет назад

what the purpose of the rocking platform

@antoniofernandes7816 4 года назад

I'm not sure but it might be to maintain the piece covered but with low quantity of the solution used

@calebm9000 5 лет назад

Why did I go to grad school there are like 50 steppppSSSS

@sssaq 7 лет назад

What is the role of (HRP) in the process of western blotting?

@ashleyseal1200 7 лет назад

HRP (horse radish peroxidase) is conjugated to the secondary antibody and when the membrane is incubated with a substrate, the proteins of interest can be detected. Some secondary antibodies are conjugated to a florescent molecule, in which case there is no need to incubate with substrate. Or you could just google it.

@sssaq 7 лет назад

Thanks. Biochemistry is very complicated, specially if you're not taking it in your first language!

@ashleyseal1200 7 лет назад

Hope I helped! Take a look at the abcam western blot video (very straight forward) or have a look on abcam.com if you're still struggling

@sssaq 7 лет назад

BioRadLifeScience Thank you very much indeed.

@ringhoilalchorei2338 3 года назад

He incubate the blot with primary antibody for 15 minutes...can we incubate for longer period like overnight at 4c

@kundansolanki33 2 года назад

For sure

@user-lp7hl6lx1n 3 года назад

Thenk you ,l m from in algeria l want dot blot steps

@luyanzhang6806 4 года назад

Why do we need two antibodies? (sorry for this dumb question, im just getting into research)

@BioRadEducation 4 года назад

Hi, Luyan! The first antibody, called a primary antibody, detects the target of interest (ex. rabbit anti-GFP). The second antibody can amplify the signal and, by more generically targeting the species that generated the antibody (ex. mouse anti-rabbit), is most cost-effective than generating reporter antibodies specific to a particular target. Visit www.bio-rad.com/classroomresources for FREE animations on how ELISA works - western blotting works because of the same target and signal amplification principles.

@suprabhagulnar5209 4 года назад

Preparation of the sample is not included?

@malithidesilva6727 4 года назад

Why do we need to add substrate?

@annakrahbichler 4 года назад

the secondary Anitbody is marked, with HRP (Horseraddish Peroxidase), to gat a signal you need to add the substrate

@jjlee784 4 года назад

유투브 알고리즘 무엇...

@heavendenies3959 5 лет назад

Most disgusting sandwich I've ever tasted.

@jatnarivas8741 3 года назад

If you tried this one, you should really try the sandwich ELISA XD

@jamshedkhan1333 4 года назад

Thank you this vedio

@AgenteWasla 3 года назад

hola chicos si estáis viendo esto suerte el viernes en TMB

@luyanzhang6806 4 года назад

can someone explain to me the theories of each step (why we do what we do)?

@BioRadEducation 4 года назад

Hi, Luyan! There is a lot to explain in a reply. You may want to check out our textbook "Biotechnology: A Laboratory Skills Course" available in digital and print formats for the science and theory of western blotting and other lab techniques. Visit www.bio-rad.com/textbook for more information.

@sachinrana1627 4 года назад

Infibulation is necessary? Why

@Jamieishere1 9 лет назад

*Strange that you place the nitrocellulose in with the gel while electrophoresis occurs. I'm not familiar with the technique... but:* 1) If the proteins can electrophoretically move along the nitrocellulose, why bother with the gel at all? 2) If the proteins can't electrophoretically move along the nitrocellulose, then surely they must blot in to it and stop migrating throughout their elecrophoretic journey, which would produce streaks of misplaced proteins.

@Jamieishere1 9 лет назад

***** Thanks for the clarification. I must have not watched the video thoroughly enough. My mistake.

@tony232cool 4 года назад

electrophoresis with gel only and then electrotransfer with gel ad membrane.

@lincolnoliveira3041 3 года назад

Spr

@ricqin8405 4 года назад

One of the worse nightmares of basically every molecular biology researcher...

@monielhashmi2402 3 года назад

Blocking solution is milk

@AnjuVerma-mz2rx Год назад

I am here , for my exam preparation ..

@Kingg_45 Год назад

Which exam

@AnjuVerma-mz2rx Год назад

@@Kingg_45 3rd semester biotechnology exam...

@laurabaigorria7029 Год назад

EL RODILLITO ME CAUSO GRACIA sjjsjs

@ahmadalansari4908 8 лет назад

@Muuip 3 года назад

An Excel file with the sequential steps and links to the video timepoints should be added as downloadable link

@spagetti001 3 года назад

is it so much to ask that you put a goddamn + and - on the anode and cathode side, instead of saying "color coding to ensure proper orientation" in the manual but never actually explain what the color coding is?

@marios1861 3 года назад

the color coding is pretty uniform. red for anode and black for cathode. sometimes black can be white or yellow or green but red stays pretty much the same.

@alecjones4676 5 лет назад

8:10 Yeah, how about we don't pour $200 down the drain...

@mitylene_bailey 5 лет назад

What should you do with it?

@jatnarivas8741 3 года назад

@@mitylene_bailey I read comments of people sealing it and reusing it at another time.

@sabrinamcalister200 Год назад

Meeting Dr Igudia RU-vid channel was the beginning of a new life for me after using his herbs medication in curing my Genital herpes Virus

@alto3218 3 года назад

や11

@earlrussell1026 4 года назад

You must love Jehovah your God with all your heart and with all your soul and with all your mind and with all your strength. You must love your neighbor as yourself. Jesus the anointed is Lord! Repent and be baptized and believe the Gospel. Work out your own salvation with fear and trembling.