To determine the isoelectric point of a protein, first establish that a gel:
(a) Contains a denaturing detergent that can distribute uniform negative charges over the protein’s surface.
(b) Exhibits a stable pH gradient when ampholytes become distributed in an electric field.
(c) Is washed with an antibody specific to the protein of interest.
(d) Neutralizes all ionic groups on a protein by titrating them with strong bases.
Correct Answer:
(b) Exhibits a stable pH gradient when ampholytes become distributed in an electric field.
Explanation:
The isoelectric point (pI) of a protein is the pH at which the protein has no net charge and, therefore, does not migrate in an electric field. To determine a protein’s pI, isoelectric focusing (IEF) is used, which separates proteins based on their charge properties in a stable pH gradient.
How Isoelectric Focusing Works:
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Establishing a Stable pH Gradient:
- The gel used for IEF contains ampholytes, which are small, charged molecules that establish a continuous pH gradient when subjected to an electric field.
- The low pH (acidic) region is at one end of the gel, while the high pH (basic) region is at the other.
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Protein Migration and Focusing:
- When proteins are applied to the gel and an electric field is applied, they migrate toward the electrode with the opposite charge.
- As a protein moves through the gel, it encounters different pH regions.
- When it reaches the pH equal to its isoelectric point (pI), it loses its net charge and stops moving.
-
Separation and pI Determination:
- Each protein becomes focused into a sharp band at its isoelectric point, allowing precise determination of its pI.
- Proteins with different pI values separate efficiently within the gel.
Why Other Options Are Incorrect:
- (a) Incorrect: SDS (Sodium Dodecyl Sulfate) is a denaturing detergent used in SDS-PAGE, not IEF. It gives proteins a uniform negative charge, which prevents pI-based separation.
- (c) Incorrect: Washing with antibodies is part of Western blotting, a technique used for protein identification, not pI determination.
- (d) Incorrect: Titration with strong bases does not establish a pH gradient for isoelectric focusing; it simply alters the overall charge state of a protein.
Conclusion:
IEF is a highly effective technique for determining the isoelectric point of a protein. By creating a stable pH gradient using ampholytes, proteins migrate to and remain at their pI, allowing precise separation based on charge.
62 Comments
Suman bhakar
March 3, 2025Done sir
Arushi
March 3, 2025👍👍
Prami Masih
March 4, 2025Okay sir
Sumit Sultan
March 6, 2025✔️✔️👍
Anita choudhary
April 18, 2025Done sir 👍
Seema
August 18, 2025D
Anita choudhary
April 19, 2025Done 👍
Neelam Sharma
August 14, 2025Exhibits a stable pH gradient when ampholytes become distributed in an electric field.
Santosh Saini
August 14, 2025PI= PH at which net charge is zero , then IEF is used for separation of protein on the basis of iso electric point or charge in stable ph gradient
Anurag Giri
August 14, 2025Ans c positive and negative dono required for separation
Aman Choudhary
August 14, 2025In IEF the protein separation is based on the charge
Karishma Don
August 14, 2025Proteins are separated according to their isoelectric point (pI). The pI depends on the balance between positively charged groups (like –NH₃+) and negatively charged groups (like –COO-) in the protein. In a pH gradient gel, proteins migrate until they reach the pH that equals their pI, where net charge = 0.
Dharmpal Swami
August 14, 2025Option c is write answer
Mitali
August 14, 2025Correct Answer:
(b) Exhibits a stable pH gradient when ampholytes become distributed in an electric field.
Khushi Agarwal
August 14, 2025I hv doubt
Urmi sisodiya
August 14, 2025Option d is correct bcoz it IEF seperate protein on basis of their isoelectric point
Varsha Tatla
August 23, 2025pH
Soniya Shekhawat
August 14, 2025IEF is separate protein on the basis of their PH so 4th is correct option.
Khushi Mehra
August 14, 2025Option a and b are both correct statements
Then correct answer is 3
Priyanka choudhary
August 14, 2025The right answer is option d,ph because of this isme net charge 0 hota h
Roopal Sharma
August 14, 2025Done
Monika jangid
August 14, 2025Ief separate protein based on their charge properties so option b is right
Surbhi Rajawat
August 14, 2025IEF focuses on separation of protein on the basis of charge hence the answer is C
Dipti Sharma
August 14, 2025Answer is C
Shivani
August 14, 2025In IEF sepration of protein is based on charge so option C is correct
Priya khandal
August 14, 2025Both a and b
Ankita Pareek
August 15, 2025Option c is correct in ief protein is separate on the basis of charge
Kajal
August 15, 2025IEF separate the protein on the basis of their PH
Kavita choudhary
August 15, 2025Answer is c
Anju
August 24, 2025Answer c
shruti sharma
August 16, 2025option b
Deepika Sheoran
August 17, 2025IEF separate the protein on the basis of their ph.
Sneha .......
August 17, 2025IEF separate the protein on the basis of PH
Shrddha Modi
August 18, 2025B on the basis of ph gradient
Bharti Yadav
August 24, 2025In IEF separation of proteins is based on charge .
MOHIT AKHAND
August 24, 2025B is correct for it 😁
Anil Choudhary
August 24, 2025C correct 💯
Priya dhakad
August 24, 2025IEF is separate protein on the basis of PH so 4th is correct option.
Bhavana kankhedia
August 24, 2025positive and negative both are required for separation
Niti Tanwar
August 24, 2025Ief is separate protein based on pH
Right is d
Alec
August 24, 2025in IEF separation of proteins is based on charge so correct ans is c.
Anjali
August 24, 2025Ans. C is correct
Sonam Saini
August 24, 2025B is right
Sakshi
August 24, 2025Exhibited stable pH gradient when amphlites becomes distributed in an electric field
Shivani Panwar
August 24, 2025B is correct option..
Devika
August 24, 2025Isoelectric point is based on ph
Falguni Prakash
August 24, 2025C on both the charge
Kashish yadav
August 25, 2025Ok sir
Ayush Dubey
August 25, 2025Exhibits a stable pH gradient when ampholytes become distributed in an electric field
sakshi khatri
August 25, 2025done
Vijay kumar Meena
August 25, 2025C ✔️ 👍
Pratibha Jain
August 30, 2025Correct answer is option (b)
Exhibits a stable pH gradient when ampholytes become distributed in an electric field.
Shriyanshi verma
August 30, 2025Exhibits a stable pH gradient when ampholytes become distributed in an electric field.
anjani sharma
August 30, 2025Answer c
IEF focuses on separation of protein on the basis of charge
Swati Choudhary
August 31, 2025IEF seperates protein on basis of both relative content of positively charged groups and negatively charged groups.
Aartii sharma
September 1, 2025IEF seprates the protein on the basis of charge
Shobha Kanwar
August 31, 2025IEF focuses on separation of protein on the basis of charge
Anisha jakhar
September 3, 2025Option b. Exhibits a stable pH gradient when ampholytes become distributed in an electric field.
Muskan Yadav
September 3, 2025IEF separate the protein on the basis of their ph
Kirti Agarwal
September 8, 2025PH
Aakansha sharma Sharma
October 1, 2025IEF is separate protein on the basis of their PH so 4th is correct option
Harish Kumar
November 18, 2025Option d is correct