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

This is what you need to know about Western Blotting. The crash course.
Western Blotting is a method for analysing proteins, used by biologists.
I introduce western blotting figures, and an example experiment to understand how to read bands.
Pop any questions you have in the comment section and I'll do my best to answer them. BUT- disclaimer- I'm just a grad student with a few years of hands-on experience & I'm not an expert.
Want to talk science with me?
/ emmaissandy
On this channel I talk about life as a PhD student in Scotland and other things. Check them out!
Pictures taken by Carmen Espejo Serrano & video by Ana Boavida. Diagrams made using Biorender.
#Biology #WesternBlotting #BiologyMajor #Immunology #CrashCourse #Cancer #CancerBiology

[@livinghope8039]

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

[@sophiaharley8276]

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

[@s.myousufuddin8608]

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

[@deanortiz1982]

Incredible video! Thanks for sharing this information!

[@wycliffenyandika9017]

thanks for the explanation, hope you have more like this

[@twistingchart3262]

incredible explanation

[@guillermogamez8222]

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

[@kamilaalicja2744]

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

[@justinpressley9807]

This was extremely helpful. Thank you so much!

[@vcrz490]

Amazing video. THANK YOU ❤

[@christinacopeland7890]

I can’t thank you enough 💜

[@StoutProper]

Absolutely brilliant video. Thank you

[@nbent4607]

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

[@MastahKitteh]

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!)

[@EmmaSandy]

Oh dear! I'll write proper CC's and sort that out 😂 thanks for the heads up.

[@isabelmelo2969]

this was so helpful! thank you so much

[@EmmaSandy]

So lovely to hear it was useful!

[@slonismo]

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?

[@EmmaSandy]

Yes- exactly that! Adding on post translational modifications changes the molecular weight and properties of the protein.

[@EmmaSandy]

Glycosylation can add vast complex strings of sugars onto the protein. These are often heterogeneous. So the vast array of (all slightly differently) glycosylated proteins in the sample gives the smear. Phosphorylation is a very small modification. Only adding ~1kDa weight to a protein. Most gels/blots can't resolve this detail.

[@slonismo]

@@EmmaSandy thank you so much! That makes perfect sense. I had the same idea but I wasn’t 100% sure. Thank you again Emma

[@jeanettespiteri7180]

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? :)

[@EmmaSandy]

Hi Jeanette, I'm happy the video was useful! If the 2 new bands are very similar in molecular weight to the protein you're blotting for, the treatment could be inducing post translational modification of the protein. Maybe the two distinct bands are new phospho-isoforms of your protein? Search 'phospho isoforms western blot' to check out some examples. If there are phospho-specific antibodies to try that could be interesting! Also you could search for 'ubiquitin ladder' for the hallmark of how ubiquitination appears on western blots. P.s. I'm also assuming your antibody is specific, these are not non-specific bands appearing, you blocked the membrane well and had clear controls! Good luck with your blots!

[@jeanettespiteri7180]

@@EmmaSandy thanks! I'll have a look at your suggestions. The antibodies are specific and the aim was to look at post translational mods following dose dependent verapmil treatment. I calculated the 2 new band's to have an approximate added Mr that would be the equivalent of polyubiquitination. The control wells showed no contamination and the 2 new band's are for 2 of the 4 sp3 isoforms. I'm getting there. I'll refer to the suggestions you made too 😊 your reply is much appreciated.

[@sarahalqithami2249]

nice explanation

[@bytesizebiotech]

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!

[@thefenerbahcesk4156]

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.

[@ahmetates893]

hocam siz hangi labda çalışıyorsunuz

[@joaopauloferreirarodrigues5576]

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!

[@PeteJudo1]

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

[@EmmaSandy]

Glad it was helpful Pete! &thanks for the shout-out 😄

[@judybohnert1380]

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!

[@manuelpv7894]

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.

[@williamarchacki571]

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

[@ismailselimyldrm]

can you share your transfer buffer recipie

[@hayaali4815]

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?

[@acru518]

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

[@EmmaSandy]

I prefer image studio lite, but I'm sure it can be done on imageJ.

[@ibsy809]

You should become a lecturer

[@thanushachisholm5807]

Would you be able to make one of coimmunoprecipitation please?

[@EmmaSandy]

Great suggestion. I will work on a video for this topic. Thanks Thanusha.

[@alidehghani2678]

clear explanation

[@casualcasual1234]

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

[@vimalmaurya7419]

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

[@irfandj100]

Thank you 👏👏👏

[@razasyed575]

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

[@MA-yt4xf]

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

[@sophietho8545]

Thank you so much, you saved my day!

[@paryamin9865]

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

[@korhanaydemir8366]

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

[@abelbabel8484]

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

[@senyobotchie4537]

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!

[@EmmaSandy]

What perfect timing, Senyo! My pleasure & I hope you're keeping well😄

[@minaakrawi6486]

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!! :)

[@EmmaSandy]

Best of luck & sending you good vibes for the MCAT ✨️👌

[@sharpandshort]

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.

[@mahmad1273]

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

[@Eunos_FD3S]

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

[@leokris7373]

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

[@sarahdisorbo9279]

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

[@virgirma1328_PhD_scholar]

Perfect explaining Dr !Thanks you very much!

[@CF-sz9fm]

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

[@joyortuno8070]

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

[@shaireenimran9442]

Thanks i am writing my thesis that helps so much

[@Tennyhu]

Amazing explanation

[@EmmaSandy]

Glad it was helpful!

[@Alex-nt1lp]

Thanks a lot for your video!

[@christandme1372]

Thanks for the help with my homework!

[@calebboyer6262]

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

[@_.jawaii._]

What does v mean? is it a control?

[@incomestockinvesting5626]

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

[@inmotion-foodco4305]

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.

[@EmmaSandy]

Hi, feel free to send me an email at [e-mail] and hopefully I can help.

[@r_pydatascience]

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

[@EmmaSandy]

Glad it was helpful!

[@Lozlaloser]

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

[@KC-hd3wi]

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

[@videobyredjade]

Thank you

[@supercriticalsolid]

this vid gonna blow up

[@inger-marieelbers8875]

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

[@EmmaSandy]

Hi Inger-Marie, have a look for 'ubiquitin ladder'. Ubiquitination typically has a smearing-ladder appearance that is quite distinct.

[@williamsutherling5881]

Excellent, concise explanation. Thanks.

[@Paprika526]

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

[@veterinarytechnology267]

Thank you prof. for your clear explanations

[@manueldelagarza2518]

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

[@EmmaSandy]

Glad to hear the video was useful Manuel! All the best.

[@alabibabatunde9711]

Thanks a lot for this

[@timmysamecphd]

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

[@asifshahriar2380]

Excellent video

[@sanjaisrao484]

Thanks

[@ivory7777]

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

[@RandomNooby]

Thank you...

[@yarenkahraman9544]

Thank you!!!

[@atrimdas1534]

amazing

[@acxelcruz710]

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

[@sophiesscience3816]

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

[@EmmaSandy]

Thanks Sophie 👩‍🔬 I'm nearly three years in!

[@LilBrownieD]

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

[@chandanar1854]

Great video and nice explanation. Thank you!

[@arduh]

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

[@carysmainwaring]

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

[@Jaziiie1]

very useful, thank you for the video