A full explanation about Bradford assay, Coomassie Brilliant Blue and the calibration curve

A full and clear explanation of the bradford assay, the coomassie brilliant blue and the calibration curve.
Here is the link of a previous video to understand the isoelectric point: • The principle of 2D Ge...

[@superbscientist743]

3:44 Ma'am you already told that the "unstable" form of CBB dye is "cationic" in nature. So how come it bears a "negative" charge ? And also, after the "donation" of lone pair to the protein, there should be "positive" charge on CBB. How come it's a negative charge ? Please help me understand. Thanks.

[@cosmicconstellations511]

I too have the same doubt. I will want someone to clarify this for me.

[@crayversace2804]

CBB is cationic in nature, bearing a positive charge, and after it donates its electron pair, it still has a positive charge, but the protein is negatively charged and thus attracted to the CBB. Makes sense?

[@MK-bx1fv]

Me too having the same doubt

[@MK-bx1fv]

@@crayversace2804 yes thank you..but in the video why does it say protein is positively charged? It would be very helpful if you could explain

[@billanthony8230]

Thank you for explaining something in 10 mins that the lecturers couldn't in 2 weeks. Appreciate it!

[@biomedicalandbiologicalsci4989]

hahaha ... thank you :)

[@jackysub1941]

at abour 3:58, why is the unstable CBB negatively-charged when it gave up a lone pair? And why is the unstable protein positively-charged when it received a lone-pair?

[@samarnasera8121]

Because when you give a lone pair, you lost it. this is why you have the negatively charged And when you receive a lone pair, you have an excess So you have a lot --> positively charged

[@s0tr88]

to continue : both are unstable. :D​@@samarnasera8121

[@ZohiraTalks]

@@samarnasera8121 dude, it's an electron, which is always negatively charged. so, when you receive it, you must go negative and when you give it, you go positive. I think she might have confused some stuff. But I ain't sure neither.

[@akamichels]

Possibly protein has sufficiently large positive charge in acidic medium, such that gaining single electron pair does not make much difference overall.

[@cosmicconstellations511]

@@akamichels I too think the same. What else can explain that the protein even after recieving a lone pair of electrons still remains positively charged.

[@Molecular_Machine]

One of the best explanations of the Bradord Assay around, great work. Keep it up

[@charlielee9774]

hi, I want to know why after CCB donate a lone pair it turn into negative charged, shouldn't it be more positive after electrons are removed? Also, why CCB is both cationic and negatively charged during the binding process, isn't these two adjective contradictory? 😵‍💫

[@Paul-su7sb]

nice video, thank you very much and I have to ask something, when the protein gets the electrons of the cbb it has to be negative and not positive or am I wrong?

[@ayselsozer67]

this is how many times she said "ok" in one video. such a helpful video though

[@nonstopnonsens2241]

Why is the unstable "Cationic" CBB negativ? A Cation is positiv i think?

[@FunnyVideos-fq8tp]

i hope she meant a reading of over 1.0 is bad not 0.1!!!

[@apollosniece9937]

I know I am probably a little late with this video but I got a question: From 08:18 on you say that we add 2 micro liters in the 4 ng/ml cup... what does the 4 ng/ml define ?

[@markkeith9055]

I thought the range for Abs was 1.0 - 0.1

[@camilojv2472]

Once I have done a bradfort assay with my protein, could I use it to do a SDS PAGE analysis

[@s0tr88]

I've my own view about this reaction which makes (for me ) more sence, added a source of acid to a diluted protein, now the (Nh2) or peptide (Nh) can have extra H, this makes protein (+), now added this coomassie which has electron pair(-), now the electron pair(-) will pool the extra H(H) from (NH3+,NH2+), the pic in 4.30 will be more sence now, lol. anyone agrees give me like :D.

[@shashankyadav6093]

How come Coomassie Blue still have -ve charge, even after donating the lone pair to Protein molecule!?

[@faizakashif9526]

how is the CBB in 3:46 negatively charged when it just got rid of a lone pair? wouldn't that make it positively charged?

[@sergeygorelik6569]

Should it be the other way around, protein is cationic because it is + and cbb is anion because it is -? A it ocnfusing

[@vedatatici9442]

by the way, I hope that you could make a video about BHP and Bana assays which are absolutely paramount in terms of purify proteins

[@alexandralynn186]

THANK YOU. I have a formal report due and I did not understand a thing until I watched this. Very straightforward. You just saved my life

[@biomedicalandbiologicalsci4989]

WOW, Thank you .. this makes me happy :)

[@chrischukwuma6469]

Cationic should mean positively charged right?

[@نسترنفریدی]

Why we first add ethanol then phosphoric acid to preparing Bradford reagent ?

[@shalyMelb]

thanks a lot

[@jahnabigoswami9713]

how can a cationic substance be negatively charged?

[@siddharthrai1327]

After giving lone pair how CBB is negative and protein positive?

[@taehyunjang9124]

I do not really leave a comment but I have to now. Thank you for the proper lecture, understood it at once. Your voice was absolutely clear to understand and have subscribed to your channel and given you a thumbs up!

[@kjeyaprakash2638]

make sure that sensitive of spec becomes less if od value goes beyond 1.0, not 0.1

[@bobexass]

Indeed, because then nonlinear processes start to occur if 1 OD gets exceeded. Nevertheless, if you have your calibration curve exceeding 1 OD and still you're lucky to maintain linear dependency, then you can count on the results since the calibration curve sums everything up.

[@tarekkollmetz8305]

Is the Bradford assay really suitable to quantify protein concentrations in the low ng/mL range?

[@brbuche]

No. UV detectors have plus or minus 0.02 AU abs fluctuations in general depending on wavelength.

[@tranhazel7944]

best explanation. Appreciate it!

[@DreadzillaLLC]

Im sorry but she sounds like dexter to me :3

[@drsirgay9176]

thank you for the great explanation

[@juliocesarrd566]

Que explicación tan clara! Muchas gracias!

[@Mohitthakurofficial]

Excellent explanation. Keep making videos like this. Make one on Lowry Assay as well.

[@biomedicalandbiologicalsci4989]

Thanks ... I will try to make a video on Lowry assay. Keep around to see all the new videos ;)

[@Mohitthakurofficial]

I'm waiting for that.

[@carlypenaemprende]

EXCELENT explanation! Thanks a lot.

[@biomedicalandbiologicalsci4989]

You are welcome ... stay tuned :)

[@hamzabassam5328]

Thx you helped me so much ☺️☺️

[@nimrahali2071]

Outclass and outstanding

[@superbscientist743]

10:08 Normally absorption should not be more than 0.1 ?? Or it should not be more than 1 ? Please clarify.

[@sogazelle]

Excellent...Merci beaucoup!!!!

[@Devyncringe]

Great explanation, I have a quiz on this tomorrow so this was very helpful, thanks!

[@Time1070]

You're one of the best demonstrators i've ever seen, I'm soooo curious about where you from, your major and what college are you in ...etc.

[@Chitario]

Why do i get a curve instead of a line? I have increasing concentrations of BSA (10ng/ul, 20, 30, 50, 75, 100) while the OD at 595nm falls with increasing concentration. Resulting in a falling curve instead of a rising straight line... The color of the wells matches the concentrations since it gets from dark red (just water) to a bright blue (highest BSA concentration). The pipetting was done in duplicate by 2 people so it cant be an error

[@alamjamal2520]

Very informative

[@ayaqz3144]

thank you miss ....?

[@superbscientist743]

Also I've a question that do we add the "unstable cationic (reddish) dye" during the assay ? How do we store this reagent in its unstable form in the lab to use in tge assay ? Or do we use the stable form ?

[@maranemonamusic6888]

I don't get it. How can it be cationic if it has electrons to donate? And if it gives electrons, how can it get negative charges and the protein who receive the electrons positive? It doesn't make sense to me, can anyone explain it please?

[@biomedicalandbiologicalsci4989]

The CBB has many groups on its structure. In an acidic medium, all the three nitrogen atoms in the CBB are protonated and so they carry a positive charge, and the two sulfonic acid groups are negatively charged, so the whole molecule will carry a net positive charge. However, the molecule in this stage can still give a lone electron pair to the protein. When the molecule gives a lone electron pair to the protein both of them become unstable and they bind to each other by ionic forces coming from the electron pair being given to the protein making the CBB electronegative and the protein electropositive in the dipole moment. The interaction then is strengthen by the VDW forces. It is a very specific and complicated chemical reaction. I hope I answered your question.

[@maranemonamusic6888]

Yes you did! Thank you very much!

[@sabnym3308]

from where exactly is the lone electron pair coming from? and why is it getting unstable just because it gives away the electrons? why should it give the electrons away and the protein takes them --> they both get unstable through it, so why do they want to get unstable?

[@vaishalithukral9129]

@@biomedicalandbiologicalsci4989 any substance that donate electrons is electropositive not electronegative. ur explaination confused me.

[@zehrayilmaz7654]

its a great video that explains it clearly. Thank you very much!!

[@franzi539]

Is it correct that coomassie Blue would interact with ANY Protein in my solution no matter if I have more life or dead cells ? So it cannot give me a quantification of the amount of viable cells in a solution right? Because cells are all lysed and then the amount of proteins are determined... Maybe someone can give an opinion. Thank you :))

[@sangeetajangra4265]

And is there any surfactant which is compatible with Bradford assay.

[@santoshsurve5289]

After giving lone pairs of electrons of CBB to proteins the CBB gets electron deficient so it should be a Cation sphere not an anion sphere , [pls comments on this ?

[@thebiobuddy9595]

You are amazing

[@satabdimandal9027]

If we had to go to other video, went tf did you make a video... have coincidence in yourself... you explain quite well

[@Trendinglive]

Nice work. Thank you for the video.

[@mastan775]

Thank you for ur simple & excellent explanation.

[@mariateixeira6136]

How can I use this video as reference? Very well explained :)

[@shubhashakya3000]

Good explanation. Thanks

[@rachelc8368]

Thanks for making this, I'll be doing this experiment today and you've made it clearer

[@demimarieowen-bowyer5629]

why is it less precise when you have an absorption reading above 0.1?

[@rachaeljones1562]

Subscribed! Can't wait to see more videos

[@biomedicalandbiologicalsci4989]

Thanx ... there will be always new videos :)

[@muhammadjaber2272]

Thanks my sister thanks my sister thanks my sister thanks my sister thanks my sister I have no words for you

[@shreshthasaxena_21]

Thank You ma'am, Great explanation!

[@sandycorn1017]

Thank you so much, this really helped

[@mohsnaziz5392]

thank you so much for explaining

[@VijayRampurkar]

really excellent ...................

[@lorenzoemanueletomasello215]

Thanks!

[@tasfa10]

In the end, when you mix the unknown protein solution with the coomassie brilliant blue and you get the concentration by measuring the absorbance, don't you get the concentration of the solution diluted in the blue? Wouldn't you have to multiply that concentration by the dilution factor to figure the original concentration?

[@arsyusuf526]

don't need to, i think... because we compare it with the dilution of BSA in standardization curve

[@khoirunnissaamini9637]

Thank you

[@Animetube-se4er]

Iiserb

[@thesublimeu1532]

very well explained...practical approach

[@sangeetajangra4265]

Mam,Thank you so much for this nice explaintion. Can you explain ,why are you saying Cationic state unstable and why anionic state is stable.With respect to which you are making this statment.

[@cosmicconstellations511]

Please keep uploading more videos! I would love to understand more on golden gate cloning and other cloning techniques.

[@steffaniadelarosajaramillo6499]

Gracias, me ayudo mucho

[@anooshkaavasare3043]

Very good explanation ma'am.

[@hosamdello2281]

Great work! I wish you could make more videos

[@ZohiraTalks]

Helped to clear things up! thanx a lot though))) keep going!!!

[@lazeenaahmed6964]

This is everything we needed thanx

[@morden1017]

May God bless this woman and channel that are more helpful than the professors who get paid to teach us.

[@noneofyobusiness9794]

Do you make serial dilutions of the unknown protein too? Or just BSA?

[@drudszzz]

Just BSA to make the standard curve. After that you will have the equation to measure the concentration of your protein of interest

[@dcdc8329]

Excellent; A++

[@alejandroe.zunigasanchez259]

Learned all this in 1 hours thanks to this video

[@2009yaoi]

Thank you !!!Easy to understand! It's very clear

[@knowledgehunter6539]

Thank you very much

[@emanmabrouki4395]

Thanks you for the excellent explanation 👍

[@vedatatici9442]

I really enjoy watching your videos they are definitely amazing.

[@shezamshazam9345]

Thank you! I hope you can upload a lot more techniques, these are proteins assays, i would love to find in your channel sugars and so on :3

[@nava9161]

Thank you!

[@tehPayett]

very well explained thanks

[@emersoncapiolos4129]

what is its iriginal color

[@felixtownsend1460]

Thank you so much!

[@adesinaomoloye7510]

very helpful clips. Thank you

[@mahmoudtolba8832]

I wish if you kindly make a video a bout "reference gene" or" housekeeping gene" used in rtPCR ... it will be so helpful as u have a great simple way of explanation... thank you

[@biomedicalandbiologicalsci4989]

Housekeeping gene is a part of the rt-PCR protocol, you use it to ensure that everything is right in your rt-PCR process ... So for example, if you are comparing 2 cell cohorts of the same cell line, in one of them one protein is inhibited (protein A) and in the other one the protein is not inhibited ... then you want to study the effect on the inhibition of this protein on the expression of a certain gene (Gene B) ... to do this, you perform rt-PCR for gene B and for another gene (Gene C). You are sure that the expression of Gene C is not affected by the inhibition of protein A. If you see any difference in the expression of Gene C between the two cohorts then you know that there is something wrong in your rt-PCR process. This is an overview, if you cant get it, I will do a video about this.

[@mahmoudtolba8832]

"If you see any difference in the expression of Gene C between the two cohorts then you know that there is something wrong in your rt-PCR process" why is that ?? what I get is that the ref.gene is the gene that is not affected by protein inhibition so it transcribe normally into mRNA but the sample gene is affected by protein inhibition then it will give me a peak that shows if it isover expressed or down expressed so we can call housekeeping gene as a blank or standard used by the researcher to compare the sample gene according to its expression

[@ericchang6388]

Liked! It helped explain a lot!

[@sabnym3308]

hi, thanks for the video. for better understanding I have some questions on it: 1. could you explain the reaction of cbbg and a protein on a molecular level, with the exact chemical reaction between the atoms? which part of the cbbg interacts with which part of the protein ( structure formula). I didnt find it anywhere in the internet so maybe you could help out. 2. why is the color chaning into blue? again: whats the chemical reaction behind it? and 3. why is the absorption higher than 0.1 not precise anymore? thanks y lot for your answers, kind regards

[@biomedicalandbiologicalsci4989]

Hello ... Thank you for your questions. 1- Actually it is so hard to describe exactly what is going on on the molecular level but I will try to facilitate the thing for you, the protein structure is very complicated and it has many side groups on its amino acids, some of these groups are ionizable and so they can accept an electron pair which comes from the CBB structure, probably from the sulphate group (although I am not sure), this donation will change the tertiary structure of the protein making it unstable (you need to imagine the 3D structure of the protein) this will enable the CBB to bind to the protein's carboxyl groups by VdW forces and to the amino groups with electrostatic interactions. 2- The color is changing because of the shift in the light absorption between the two states of the dye resulted from the change in its chemical structure. 3- The absorption vs. concentration curve is linear when the protein concentration is not too high, but on a certain point when the protein concentration is too high the curve enters a steady state because there will be no more CBB molecules in the solution to interact with the protein molecules so the absorbance will not increase anymore. Due to this, if you get an absorbance that is higher than 1.0 you should dilute your sample.

[@biomedicalandbiologicalsci4989]

I hope I made it easier for you to understand :)

[@xzlatan9257]

Great explanation.

[@shwethaliyanage6804]

Thank you for the explanation

[@tashanicole1369]

You were so thorough. Thank you!

[@mohammedalmdr4209]

Very very very good ❤❤❤

[@erengulumser2414]

I LOVE YOU!

[@shivshankarsaha5812]

improve ur speaking skills plse at least for god seek!!!!

[@sadeenissa6492]

what about improving your spelling skills first, for god’s “seek”...!!!

[@drudszzz]

She speaks very well and very clearly 🥺 why are you a hater 🤧 you can do it then