Western Blotting

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Western Blotting

Рет қаралды 1,065,910

11 жыл бұрын

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.
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Пікірлер: 131

@nikkibryceroque3026 6 жыл бұрын

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

@medielijah 4 жыл бұрын

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

@resistance110 4 жыл бұрын

@@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 жыл бұрын

@@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 жыл бұрын

@resistance110 and @oscar you guys are awesome

@ronaldokevin5693 3 жыл бұрын

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

@michelebos5231 7 жыл бұрын

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 6 жыл бұрын

thnku sir

@lincolnoliveira3041 3 жыл бұрын

Yuka

@yodaydyxz1752 9 жыл бұрын

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

@starrynight7891 9 жыл бұрын

Thank You so much for your wonderful video!

@Hoxgene 2 жыл бұрын

That final result was beautiful!

@hamlilasma5434 Жыл бұрын

all respect for this valuable work , thank you 🎩🌸

@noemimirra7907 8 жыл бұрын

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

@muhammadyudadwitamaagustia9883 4 жыл бұрын

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

@rowang5578 2 ай бұрын

He uhh he give me butterfly 🤭 those green hand

@EDUARDO12348 8 жыл бұрын

Thank you BioRad

@arshisstudio1394 7 жыл бұрын

thank u so much.. this video was so helpful

@mohammedal-hammadi5085 3 жыл бұрын

Thank you so much, it's pretty helpful

@joyjustman3536 Жыл бұрын

Wow! This is so helpful I must say

@marcussmith9461 5 жыл бұрын

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 ?

@nurshahilinamira4165 5 жыл бұрын

Nice video!

@lmacunac 8 жыл бұрын

Nice video about it!

@jaimes6152 7 жыл бұрын

THANK YOU.

@tutoradvance 9 жыл бұрын

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?

@Dilmi24 9 жыл бұрын

Thanks so much! :))

@adronung1892 4 жыл бұрын

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

@laurentiaadinda8439 4 жыл бұрын

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

@monishajayabalan7823 4 жыл бұрын

You are not alone 😂😂

@user-we9qg3dy5n 3 жыл бұрын

Same

@jatnarivas8741 3 жыл бұрын

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

@HercadosP 3 жыл бұрын

@@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

@1904Aparna Жыл бұрын

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

@SaSaRush 2 жыл бұрын

Very helpful thank you

@user-rf4vc7mt4d 5 жыл бұрын

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

@user-rf4vc7mt4d 3 жыл бұрын

Scam

@davidwaziri8460 2 жыл бұрын

Vedio

@p91grwm14 3 жыл бұрын

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

@menglinzhao2536 2 жыл бұрын

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 жыл бұрын

Thanks Bio-Rad ....

@emilie6466 4 жыл бұрын

Govind Meena thanks Bio-dad...

@lamhirhcn 9 жыл бұрын

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 6 жыл бұрын

Lamhirh amen to that!

@TheTillieTube 5 жыл бұрын

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

@ShakespeareCafe 5 жыл бұрын

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 Жыл бұрын

How to recyle it??

@NewWesternFront Жыл бұрын

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

@akansha4575 Жыл бұрын

Nicely explained 👏💐thnku

@trilochans5008 2 жыл бұрын

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

@Birs_84 3 жыл бұрын

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? ^_^

@ruths6022 3 жыл бұрын

Thank you sir

@hanumakumar8997 2 жыл бұрын

Thank you

@ayeshasultana6391 7 жыл бұрын

Thanks !

@twinkle1548 2 жыл бұрын

Thank u soooooooooooo much🙏

@yifanhu1546 8 жыл бұрын

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 жыл бұрын

+BioRadLifeScience i see, thank you so much!

@tony232cool 4 жыл бұрын

how do yo do detection after electrotransfer.

@user-fn7hs5yc2h 8 жыл бұрын

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 8 жыл бұрын

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

@diogenesofsinope2692 Жыл бұрын

@@user-fn7hs5yc2h explain pls

@pinkyflowe5762 7 жыл бұрын

it helpful video. thnx

@ishaqadjaimi9645 5 жыл бұрын

Thanks

@stephenchetwynd7760 4 жыл бұрын

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 жыл бұрын

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.

@dawoodsalar7202 5 жыл бұрын

why we add scondry antibodies????

@destroya3303 8 жыл бұрын

a rocking board is that really necessary?

@ScienceGeek23 3 жыл бұрын

Cool gloves!

@jie0330 6 жыл бұрын

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

@user-bt8ru7rm9w 5 жыл бұрын

can i use salmon sperm DNA for blocking solution?

@kasraakbari229 5 жыл бұрын

wow!!!!! great

@suprabhagulnar5209 4 жыл бұрын

Preparation of the sample is not included?

@xenawu1588 8 жыл бұрын

what is the purpose of the blocking solution?

@nicheng1927 8 жыл бұрын

+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.

@DeepsikaP 2 жыл бұрын

Grt video! Can the antibodies be reused?

@puspanjali2464 Жыл бұрын

Yes you can

@JohnSmith-td7hd 6 жыл бұрын

Reminds me of developing film.

@-..l 4 жыл бұрын

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

@JM.5387 3 жыл бұрын

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

@ringhoilalchorei2338 3 жыл бұрын

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

@kundansolanki33 2 жыл бұрын

For sure

@ConCerN.exe. Ай бұрын

👍 Helpful

@luyanzhang6806 4 жыл бұрын

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

@BioRadEducation 4 жыл бұрын

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.

@pecatum666 10 жыл бұрын

Why haven't you added any blocking solution?

@sssaq 7 жыл бұрын

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

@ashleyseal1200 7 жыл бұрын

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 жыл бұрын

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

@ashleyseal1200 7 жыл бұрын

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 жыл бұрын

BioRadLifeScience Thank you very much indeed.

@geetarani2875 5 жыл бұрын

good

@calebm9000 5 жыл бұрын

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

@roseb2105 8 жыл бұрын

what the purpose of the rocking platform

@antoniofernandes7816 4 жыл бұрын

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

@user-lp7hl6lx1n 3 жыл бұрын

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

@malithidesilva6727 5 жыл бұрын

Why do we need to add substrate?

@annakrahbichler 5 жыл бұрын

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

@jamshedkhan1333 4 жыл бұрын

Thank you this vedio

@luyanzhang6806 4 жыл бұрын

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

@BioRadEducation 4 жыл бұрын

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.

@Suzi-g9f 4 жыл бұрын

유투브 알고리즘 무엇...

@Jamieishere1 9 жыл бұрын

*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 жыл бұрын

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

@tony232cool 4 жыл бұрын

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

@sachinrana1627 4 жыл бұрын

Infibulation is necessary? Why

@AgenteWasla 3 жыл бұрын

hola chicos si estáis viendo esto suerte el viernes en TMB

@lincolnoliveira3041 3 жыл бұрын

Spr

@AnjuVerma-mz2rx Жыл бұрын

I am here , for my exam preparation ..

@Kingg_45 Жыл бұрын

Which exam

@AnjuVerma-mz2rx Жыл бұрын

@@Kingg_45 3rd semester biotechnology exam...

@heavendenies3959 5 жыл бұрын

Most disgusting sandwich I've ever tasted.

@jatnarivas8741 3 жыл бұрын

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

@laurabaigorria7029 Жыл бұрын

EL RODILLITO ME CAUSO GRACIA sjjsjs

@ricqin8405 4 жыл бұрын

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

@monielhashmi2402 3 жыл бұрын

Blocking solution is milk

@Muuip 3 жыл бұрын

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

@ahmadalansari4908 8 жыл бұрын

@spagetti001 3 жыл бұрын

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 жыл бұрын

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 жыл бұрын

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

@mitylene_bailey 5 жыл бұрын

What should you do with it?

@jatnarivas8741 3 жыл бұрын

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

@sabrinamcalister200 Жыл бұрын

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

@alto3218 3 жыл бұрын

や11

@earlrussell1026 4 жыл бұрын

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.

@Verspassungsschutz 4 жыл бұрын

Shut Up