Biuret Test for Proteins | Protein assay | Estimation of Protein | Dr. Nagendra Singh | PENS#38

#biuretassay #proteinestimation #labexperiment
The biuret test is a chemical test used for detecting the presence of peptide bonds in proteins an dpolypeptides. copper ions form coordination complex with peptide groups i proteins which absorb at 540nm. The biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptides. The principle of the biuret assay is similar to that of the Lowry method, however it involves a single incubation of 20 min and takes lesser time than lowry assay.
The biuret is a good general protein assay for batches of material for which yield is not a problem. The Bradford assay is faster and more sensitive.
The main methods of protein estimation are:
1. UV based detection
2. Biuret method
3. Lowry method
4. BCA method
5. Bradford method
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[@arvindprajapati4723]

In Biuret Reagent,if we use KOH at place of NaOH then what will be effect of KOH ?

[@vishamberdayal946]

If no biuret used in biuret reagents in protein estimation .so why it is called as biuret test for protein estimation?

[@ibt012kajalyadav7]

Wht would happen if metal ion chelaters like EDTA are present in sample?

[@ibt012kajalyadav7]

Does biuret reagent react with amino acids?

[@vidurchoudhary2276]

What would happen if we use ethylene glycol instead sodium potassium tartrate (Rochelle salt) in biuret assay?

[@mulhimmukhtar1474]

How BSA concentration(mg/ml) of test tube 1-5 is calculated?

[@ericisabwa6498]

A nice presentation

[@proteinengineering007]

@ERIC ISABWA Thank you

[@mansichaturvedi6777]

Will the resultant colour vary between polypeptide (upto 50 aa) & protein sequence ( more than 50 aa)??

[@proteinengineering007]

should not...

[@ritujasingh5249]

If we increase the amount of protein in sample, what possible effect it will have on copper reduction?

[@shwetatyagi8160]

Biuret reacts with Cu2+ ions to form a violet/purple complex that consists of four biuret molecules complex with one cupric ion. The lone pairs present on the Nitrogen of the peptide linkage bonds with the cupric ions in the reagent to create a violet purple colour. if protein molecules are present in large amount then there will be more dipeptide bonds formation leading to a darker color.

[@Lavanya3000]

If we want to measure the concentration of a protein complexed with some other molecule such as lipoprotein, will this method still work?

[@ibt007gaurisingh4]

No, it won't work in that case, because biuret method is highly specific for proteins and peptides. Further if any other compound consists of nitrogen or metal ion chelators then it would react with copper ion and may cause an error in the test.

[@proteinengineering007]

yes

[@vidurchoudhary2276]

And if we take CuSO4 instead of CuSO4.5H2O? will this effect the biuret test?

[@muskaanraghav342]

CuSO4 is amorphous, whereas CuSO4 5H2O is crystalline. while CuSO4 5H2O is the hydrated form of copper(II) sulfate. This is the main difference in their nature which will not lead to the purple colour formation in biuret test therefore, CuSO4 .5H2O is used .

[@vidurchoudhary2276]

What will we observe if we reduce the alkalinity of the solution in biuret test?

[@arvindprajapati4723]

We will observe that no.of cuperic ion will going less in no.as alkalinity reduces . because alkalinity is favorable condition for formation of cuperic ion in biuret.

[@Lavanya3000]

What is the black precipitate formed when preparing biuret reagent?

[@muskaanraghav342]

The black precipitate formed is Cupric oxide.

[@kirti6753]

Is it possible to conduct biuret test to determine proteins for a wastewater sample?

[@ibt042srishtichauhan3]

Yes it is possible ,as long as the protein present if any is 3 amino acid long but as the conc is low unlikely you will see the colour change by an eye but buriet method may not be appropriate for low concentration . Consider Bradford or lawry test .

[@arvindprajapati4723]

Why biuret method is very sensitive and rapid for protein estimation as compared to others methods?

[@ibt042srishtichauhan3]

Compared to others BCA is one of the sensitive assay , it can detect protein at low concentration , it has less variability then others .

[@salonigupta9520]

Can we use biuret method to test the amount of protein in milk?

[@janvisingh3721]

Yes we can use biuret method to test amount of protein in milk as it is quantitative calorimetric test.

[@kohsheenbaliya]

Does BSA has any absorbance at 595nm in the absence of barford reagent

[@proteinengineering007]

shudnt be

[@foodtechnologist838]

Kindly reply me sir..is sodium potassium tartrate and potassium sodium tartrate same?? Because I have potassium sodium tartrate.

[@AREEBA_aa]

yes is is obvious....

[@racingcar77389]

When we do experiment of buriet test, how does color change?

[@janvisingh3721]

To find the concentration of protein in a given sample we use biuret test. The color produce when conjoint peptide bonds react with copper ions which give violet color complex.

[@mukshy_1509]

Which free amino acid gives a positive biuret test?

[@ibt012kajalyadav7]

Histidine is the only free amnio acid which gives a positive biuret test because of the absence of additional reducing agent.

[@piyushparmar8991]

The last calcution will be 17.6 because 0.5 ml of portein contain 8.8 so for 1 ml it would 8.8+8.8 so tha answer for last 1 ml of protein is 17.6. Can anyone explain me ?????

[@proteinengineering007]

Yes..its typo error..it is 17.6 @Piyush Parmar

[@piyushparmar8991]

​@@proteinengineering007 thank you sir And can you make a vdo of qualitative analysis of phytochemical I'm currently working on it I got the od but don't know how to calculate into a acutal weight of alkaloids

[@proteinengineering007]

@@piyushparmar8991 which method are u using?

[@JanhaviRai-IBT-]

Is biuret test is helpful in determining the protein for wastewater sample?

[@_ibt_AditiSharma]

No, because waste water may contain nitrogenous compounds and other metal chelators which effect the efficiency of biuret method.

[@proteinengineering007]

@@_ibt_AditiSharma true

[@amritasingh6483]

Why Biuret method is less sensitive than Lowry method.

[@janvisingh3721]

Folin reagent help in reducing copper ions in lowry method makes it 100 time more sensitive than biuret reaction.

[@karticksharma1306]

Why is the molarity taken 0.2 M and not slightly more or less?

[@proteinengineering007]

concentration of standard is taken as per sensitivity of the method

[@vidurchoudhary2276]

What is the use of potassium iodide in biuret test?

[@arvindprajapati4723]

To prevent reductions of alkaline copper tartrate and separation of cuprous oxide.

[@JanhaviRai-IBT-]

How does the biuret reagents work to detect the protein?

[@niharika4764]

A Biuret test is a chemical test used to determine the presence of a peptide bond in a substance. It is based on the biuret reaction in which a peptide structure containing at least two peptide links produces a violet color when treated with alkaline copper sulfate.

[@angelasusan8629]

bruh-

[@kitchenwithsara1]

Spectrophotometre ka b btaye standard curve protein k liye

[@proteinengineering007]

same to same

[@kitchenwithsara1]

@@proteinengineering007 mean ye he kr ly?

[@kitchenwithsara1]

Mjhy aik aur formula smjhna hao jis mai 260/280 abs wala

[@proteinengineering007]

@@kitchenwithsara1 that is used in estimation of purity of extracted DNA..The ratio of absorbance at 260 and 280 nm is used to assess DNA purity. A ratio of ∼1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower (≤1.6), it may indicate the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm.

[@kitchenwithsara1]

@@proteinengineering007 if i want to check protein purity then?

[@yubrajpani3653]

How.to calculate bsa concentration in mg/ml

[@proteinengineering007]

u can use balance to weigh BSA powder and dissolve accordingly. @Yubra Pani

[@pratyakshasingh8709]

Do all the proteins respond to biuret test?

[@janvisingh3721]

All protein do not contain the same amino acid hence do not respond to all color reactions.

[@rohantyagi9266]

Why is CuSO4 used in biuret test?

[@mukshy_1509]

CuSO4 used in biuret test because it is the main source of cupric II ions (Cu2++) in sol

[@kohsheenbaliya]

What is the role of ethanol in barford reagent

[@simranshree8004]

The role of ethanol in the Bradford reagent is to act as a reacting functional group, since the OH at the end of the ethanol/methanol can be depronated( where the H leaves the oxygen) in either slightly acidic(+H3O) or slightly basic conditions( -OH). And once the methanol/ethanol becomes depronated, another compound can be added to the carbanion.

[@shalnisrivastava7978]

Why do amino acids give a negative biuret test?

[@priyanshusharma3079]

No, The bond is formed between the α amino group of one amino acid and the α carboxyl group of another amino acid with the removal of a water molecule.The peptide bonds in Biuret give a positive result for the test. Peptide bonds are required for the formation of the chelate complex.

[@rinkukumari3380]

What is the principle of biuret method...?

[@ujjwalpriya5894]

Presence of proteins in biological fluids.

[@niharika4764]

why cuso4 is not used in excess in biuret test?

[@ritujasingh5249]

Excess of copper sulphate must be avoided in making the biuret test, since the color of the salt prevents the recognition of the color produced in the reaction. The presence of ammonium salts interferes with the test.

[@JanhaviRai-IBT-]

cuso4 in not used in excess in biuret test because the colour of the salt present in that will prevent the recognition of the colour produce in the reaction

[@niharika4764]

why would amino acid give a negative result in the biuret test?

[@pratyakshasingh8709]

The biuret test is also used to detect proteins. That is because proteins are made up of polypeptides, which in turn, are made of amino acids joined by peptide bonds. ... A negative result (lack of violet colour formation) may mean lack of protein, or the presence of free amino acids (without peptide bonds).

[@Lavanya3000]

Biuret test is for peptide bonds. As single amino acid do not have peptide bond, they give negative result with biuret's reagent.

[@JanhaviRai-IBT-]

It is because biuret test is for the peptide bond and this test is used for the single amino acids but peptide bond not form in single amino acids hence this test gives negative result .

[@proteinengineering007]

@Addhyan Negi good

[@rohantyagi9266]

What Colour does biuret turn when protein is not present?

[@pratyakshasingh8709]

There will be no color change. Blue color of biuret reagent will remain same.

[@teniolafreeman6515]

@@pratyakshasingh8709 what if it turns pink? Does that indicate a positive test too?

[@rohantyagi9266]

What does a positive biuret test indicate?

[@niharika4764]

The biuret test is a chemical assay that detects the presence of proteins in a sample. The test relies on a color change to confirm the presence of proteins. If proteins are found, the sample will turn blue.

[@proteinengineering007]

@@niharika4764 positive biuret means presence of nitrogenous groups in the sample.

[@digitalkitchenworld7520]

How to calculate conc.sir

[@proteinengineering007]

just put values in y=mx+c and calculate value of x from standard graph. thats concentration

[@pratyakshasingh8709]

Why naoh is added first in biuret test?

[@niharika4764]

The NaOH dissolves the protein so that protein can then react with the copper salts. If you directly add copper sulphate in NaOH they will react to form copper hydroxide. If you add too much NaOH, the copper hydroxide is the major reaction, and the purple colour is hidden

[@_ibt_AditiSharma]

NaOH provides alkaline conditions to the solution for the reaction to occur.

[@JanhaviRai-IBT-]

NaOH gives a alkaline condition to form any reaction in biuret test.

[@priyanshusharma3079]

What are the limitations of biuret test?

[@arvindprajapati4723]

Presensce of nitrogenous compounds and chelaters like EDTA. In sample contain any other nitrogen atom that also can reacts with Cupric ions and give some colour ,So that can give rise error in protein estimation.

[@arvindprajapati4723]

Why is biuret reagent purple ?

[@mylizardprotectsmefrombugs2568]

shouldnt it be 17.6 mg/ml at the last

[@proteinengineering007]

@Naman Gupta ....yes... it should be 17.6mg/ml.....thank u for pointing out..its a typo error...

[@hrithikchatterjee1773]

@@proteinengineering007 sir can u tell how u measure the total?

[@proteinengineering007]

@@hrithikchatterjee1773 first u calculate protein concentration in the final sample volume...eg..in this case the value of x = 2.2 mg/ml (as per standard curve)....now u see that total final volume of sample = 4 ml... that means 4 ml will contain 2.2 x 4 =8.8 mg protein....now u see, initially only 0.5ml actual protein sample was taken which got eventually diluted to 4ml.....so we can say that 0.5ml sample contain protein that is now present in 4ml= 8.8.mg 0.5ml contains- 8.8.mg 1 ml will have = 8.8/0.5 = 17.6mg so we sat that concentration of unknown sample = 17.6mg/ml hope it woud help u

[@hrithikchatterjee1773]

@@proteinengineering007 thanks sir