Western Blotting
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- čas přidán 15. 10. 2012
- 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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I only watched this because of my assignment and gosh I've never been so overwhelmed by such laborious procedures!
lab is not your metier, try something else, no shame
@@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.
@@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
@resistance110 and @oscar you guys are awesome
@@medielijah social media is not good for you, try something else, no shame
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 !
thnku sir
Yuka
Very informative
Thank you so much for taking time and explain each step.
Thank You so much for your wonderful video!
That final result was beautiful!
all respect for this valuable work , thank you 🎩🌸
Thank you BioRad
WATCH IN X1.25 SPEED just to save you some time
Scam
Vedio
when he was discarded the wash buffer or antibody, it gave me anxiety
He uhh he give me butterfly 🤭 those green hand
really enjoyed watching this video, great review!!! Thank you!!!
Thank you so much, it's pretty helpful
thank u so much.. this video was so helpful
Wow! This is so helpful I must say
"pour off the primary and secondary antibody"
me watching this: o m g, i sealed and reused for many times bcs expensive😂😭
You are not alone 😂😂
Same
Have you found the results to be equally reliable either way?
@@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
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...
Lamhirh amen to that!
This is how we do it in real life....
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
How to recyle it??
@@seetheworld6656 put it in the blue can on your house curb
Nice video about it!
THANK YOU.
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? ^_^
Nice video!
What is the composition of blotting buffer, blocking buffer, wash buffer?
Thanks so much! :))
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?
Thanks Bio-Rad ....
Govind Meena thanks Bio-dad...
Very helpful thank you
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 ?
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?
Do you use nitril gloves? Are they resistant in contact with methanol in the Buffer?
Thank you
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
Thank you sir
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?
***** Thank you for the reply, point clarified.
@@user-fn7hs5yc2h explain pls
Nicely explained 👏💐thnku
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?
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.
Thanks!!!
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.
+BioRadLifeScience i see, thank you so much!
how do yo do detection after electrotransfer.
Thank u soooooooooooo much🙏
Cool gloves!
it helpful video. thnx
Thanks
How much duration of time take to diagnose western blot test for hiv in lab???(not about window period)
it is 2018 and something has been updated ( instructions and so on)
a rocking board is that really necessary?
Reminds me of developing film.
John Smith I agree, like developing film. Expect, it is not in a dark room.
In the old days, we used photographic film, and developed it in a dark room. Everything was labeled with radioactivity.
why we add scondry antibodies????
wow!!!!! great
👍 Helpful
Grt video! Can the antibodies be reused?
Yes you can
Preparation of the sample is not included?
What is the role of (HRP) in the process of western blotting?
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.
Thanks. Biochemistry is very complicated, specially if you're not taking it in your first language!
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
BioRadLifeScience Thank you very much indeed.
good
He incubate the blot with primary antibody for 15 minutes...can we incubate for longer period like overnight at 4c
For sure
Why haven't you added any blocking solution?
Why do we need two antibodies? (sorry for this dumb question, im just getting into research)
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.
Why did I go to grad school there are like 50 steppppSSSS
what is the purpose of the blocking solution?
+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.
can i use salmon sperm DNA for blocking solution?
hola chicos si estáis viendo esto suerte el viernes en TMB
Thank you this vedio
what the purpose of the rocking platform
I'm not sure but it might be to maintain the piece covered but with low quantity of the solution used
Why do we need to add substrate?
the secondary Anitbody is marked, with HRP (Horseraddish Peroxidase), to gat a signal you need to add the substrate
유투브 알고리즘 무엇...
Thenk you ,l m from in algeria l want dot blot steps
can someone explain to me the theories of each step (why we do what we do)?
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.
*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.
***** Thanks for the clarification. I must have not watched the video thoroughly enough. My mistake.
electrophoresis with gel only and then electrotransfer with gel ad membrane.
One of the worse nightmares of basically every molecular biology researcher...
I am here , for my exam preparation ..
Which exam
@@Kingg_45 3rd semester biotechnology exam...
Spr
Blocking solution is milk
Infibulation is necessary? Why
EL RODILLITO ME CAUSO GRACIA sjjsjs
An Excel file with the sequential steps and links to the video timepoints should be added as downloadable link
Most disgusting sandwich I've ever tasted.
If you tried this one, you should really try the sandwich ELISA XD
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?
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.
X
8:10 Yeah, how about we don't pour $200 down the drain...
What should you do with it?
@@mitylene_bailey I read comments of people sealing it and reusing it at another time.
や11
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Thanks !