Western Blotting | Beginner Data Interpretation Tutorial & Step-by-Step Protocol Explained 

You have explained this better in 10 minutes than an entire module on Western Blotting at university has!! Thank you!

The loading control tells me that there is possibly no band with 0 treatment because too little sample was loaded. This blotting needs to be repeated. Great video. Where GAPDH is weakest is precisely where we don't see band or expression is decreased. Great lecture!

As a non native English speaker, I take note of "You can see the levels are not entirely consistent, this is a genuine Western Blot I did and it's not perfect. I believe this sentence will be of help to me. Jokes aside, very insightful video, I enjoyed it a lot.

You are simply BEAUTIFUL with BEAUTIFUL WORK... I Love you🤣, so I subs + liked ur videos... Thank you, Emma!! From Master's student.

yo big dawg ur actually the man helping people out, we Native Americans truly appreciate it

This is such an informative video. I just recently started doing Western Blotting and was confused about reading the blot itself, but you explained several good points that have really helped me out! Thank you so very much for doing this video!

Best explanation on the web, thank you! I've been searching far and wide for this.

Thanks so much for the clear explanations! Hope to see more videos on protocols and data analyses, it helped me a lot :)

Thanks so much! Comprehensive + concise (and it's interesting that you show why this technique matters with the example from your cancer research!)

Thanks so much for making these videos, Emma. It's nice to have a secondary resource to review these methods besides the people in your lab - especially if the people in your lab get annoyed every time you ask questions.

Incredible video! Thanks for sharing this information!

Perfect explaining Dr !Thanks you very much!

such an incredibly helpful video can't even begin to describe, thank you

Very great explanation. Very bad blot itself though 😅 A few recommendations. Some, you may already be doing: 1. BSA Protein Concentration Assay for all samples simultaneously prior to running them on a western. 2. Never run more than 15 ug of Protein per lane. If this is below your threshold of detection, switch to a commercial blocking buffer such as SuperBlock from Invitrogen in TBS-T. That should help with sensitivity. The reason that 15 ug should be the upper limit is because you have to be within the linear range of protein concentration for an antibody to be used for relative quantification. For a protein like GAPDH, this linear limit in many cells lines is 5 ug. So in addition, I would also use a different HSKG like beta actin or vinculin. 3. Instead of a housekeeping gene, you can also go with a total protein stain. You used Ponceau, but depending on the company you buy it from, it's really hit or miss. And if you make your own Ponceau as I used to, that's even less consistent between lots. Some people just forgo housekeeping genes all together and only go with the total protein stain, but it's also possible to use 2-3 housekeeping genes on the same blot so that you can take the best 2 of 3. If you have a problem with 2 of them, then you're either modifying expression of 2 of them OR your sample concentration was measured accurately. 4. if you heat your samples, don't go over 10 mins at 95C. Can cause hydrolysis of your protein and that can also effect how it shows up on the blot and potentially account for why your GAPDH isn't even. Should help you get more consistent results and thus graduate faster. Good luck!

You explain super well. This type of content is great.

Thank You For this awesome tutorial! This cleared all my confusions.

Excellent, concise explanation. Thanks.

Thanks for the video! It was really helpful and straight to the point!

this was so helpful, thank you!! other videos were super confusing but this made so much sense :)

Thank you prof. for your clear explanations

Oh wow! Emma!! I just thinking to myself the other day and was going ask if you could do a tutorial on Western Blot for beginners but you were way ahead of me on this one!!! Thanks so much!

Absolutely brilliant video. Thank you

Amazing video! It really helped me to undestand a paper i was struggling with

I found this very fascinating. Thanks for sharing Emma. :)

This was extremely helpful. Thank you so much!

thanks for the explanation, hope you have more like this

Thank you for this! I get to see the application of Western Blot!!

Amazing video. THANK YOU ❤

Thank you so much, you saved my day!

Great video and nice explanation. Thank you!

Nice explanation! It was a quick refresher to my lab courses that I did more than 10 yrs ago.

Thank you so much Emma! You have saved me as I'm studying for the MCAT. You're method of explanation made me fully understand this in 10 minutes after trying to read 3 different textbooks!! :)

you're great at explaining! thank you for this video :)

Thanks a lot for your video!

Thanks for the help with my homework!

This is such a good explanation. Thank you Emma, extremely helpful.

Thank you for clearly explaining the procedure, it really helped me a lot in understanding it.

Amazing! Thank you so much for this. Can you please make similar videos on northern and southern blotting?

Excellent video

I can’t thank you enough 💜

this is the best video on western blot. thanks...

incredible explanation

Thank you!!!

This is such a useful video, thankyou !!!!!

Thanks Emma for this nice video.✌👍 Please make a video on background subtraction in WB.

very useful, thank you for the video

Awesome video! You make it easy to understand. Also... you have such a cute accent!!

Thank you, I hope that thanks to your video I'm about to pass exam on my university lol

Thanks a lot for this

Thanks i am writing my thesis that helps so much

Thank you 👏👏👏

this was so helpful! thank you so much

Studying for mcat super helpful for bio biochem section thank you so much!!

clear explanation

Amazing explanation

This vedio is really helpful... Please make another vedio for dentiometric analysis of bands using imaj J software

Thank you...

Thank you

amazing

Such a info informative video, thank you!! Can you do one about ELISA?

this vid gonna blow up

This was a great video! Did you look at changes in the expression of P53 and/or P21? How did you determine the changes?

Great video! How do you feel about semi-quantitative densitometry after having n=3 blots? Can help to quantify those band differences!

Thanks

As CEO of blots I give this video a 10/10.

you need to be in academia! explained so clear thanks a bunch!

i tried to turn on CC and it decided you were speaking Dutch :') thank you youtube very helpful (that aside, this video helped me a lot!)

Bane of my bloody existence, western blots and live cell imaging

Many thanks, that was very good, especially your clarity, streets ahead of other WB videos. I have two questions: 1) is there a published paper (or two) that you know of where the WB are key, reveal some key insight? 2) You admit to being dissatisfied with the variable intensity of your loading control ... why did you not wait for a better one for your video? It leaves me with the impression that WBs are hard to get right .. and perhaps they are .. just wondering if that is part of your message here.

Hello, this was very concise, really really helped watching this. Can I ask what the protein CD45 is labelled at the end WB?

I need to know about BCA quantification assay. Calculations for working reagent and serial dilution with protein. How we can calculate quantity of protein in serial dilution?

Now I just need a video on how to photoshop Western Blots better than Stanford president.

Love this! Where are you at in your PhD journey?

Hi Emma, thanks for uploading a western blot video. quick question, I have used this technique on a number of muscle biopsy samples and now need to make sense of them on Li-cor image studio. Do you do freelance tutoring in this area? as I help a little help I think.

Hi Emma, thank you for sharing this in such a clear manner, my tutors have not managed to do such a good job yet! I'm currently doing an WB analyses to establish the effect of different doses of verapmil on SP3. When I run my blot for the cultured cells with treatment with Sp3 antibody I get 2 new bands. I am not sure what this indicates. Clearly the Sp3 antibody is picking up the protein but the 2 new bands are for differnet Mrs and this is before I do another run with ubiquitin. Any ideas? :)

can you share your transfer buffer recipie

Hi. I am currently investigating ubiquitination of Sp3 isoforms in Caco-2 cells. What would visually indicate ubiquitination on a WB

You should become a lecturer

Ru doing normalization using ImageJ for densitometry for your gel??

Would you be able to make one of coimmunoprecipitation please?

Thank you so much for this video! Do you think you could elaborate just a tad as to exactly WHY a smeary band indicates post-translational modification of that protein? Is it because of slight changes in the weight of that protein? How come the same is not true for phosphorylation?

What does v mean? is it a control?

nice explanation