In an attempt to detect protein expression profile in a cell, Western blot techmque is employed. Expression of two new proteins is to be followed by probing with respective high affinity antibodies (raised in rabbit). Unfortunately, the tow proteins were found to co-migrate in SDS-PAGE profile. Under this situation, using one dimensional SDS-PAGE and by western blot, which one of the following is the best way to demonstrate the presence of both the proteins?
A. Develop Western blots with their antibodies in the same gel.
B. Prior to doing SDS-PAGE/Wætem blot, one protein could be removed by immunoprecipitating in the cell extracts.
C. Silencing the expression of one protein at a time by siRNA and performing Western blotting.
D. Subjecting the technique of stripping/reprobing of the gel after transferring to nitrocellulose membrane while doing Western blotting
Western blotting is a widely used technique to detect protein expression profiles in cells. However, when two proteins co-migrate in SDS-PAGE, distinguishing them can be challenging. If high-affinity antibodies (raised in rabbits) are used for detection, the best strategy to demonstrate the presence of both proteins is:
Correct Answer: Stripping and Reprobing
Among the given options, the best approach is:
✔ D. Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Why is Stripping and Reprobing the Best Approach?
- Preserves Sample Integrity – Instead of running multiple gels, the same membrane can be used for sequential probing with different antibodies.
- Saves Time & Resources – Avoids the need for additional sample preparation and gel runs.
- Allows Sequential Detection – By first detecting one protein, stripping the membrane, and then probing for the second protein, both can be confirmed separately despite co-migration.
Why Not the Other Options?
- (A) Developing Western blots with both antibodies in the same gel: Not ideal because if both proteins co-migrate, their signals may overlap, making it difficult to distinguish them.
- (B) Immunoprecipitating one protein before SDS-PAGE: This removes the protein, but it does not allow simultaneous detection of both in the same blot.
- (C) Using siRNA to silence one protein at a time: This is labor-intensive and not necessary when a simpler method like stripping and reprobing is available.
Conclusion
Stripping and reprobing Western blots is the most efficient method to differentiate co-migrating proteins while maintaining sample integrity. It provides clear, sequential detection without the need for complex sample modifications. For researchers facing co-migrating proteins, this method is a practical and reliable solution.
53 Comments
Prami Masih
March 4, 2025Okay sir
Suman bhakar
March 5, 2025👍👍
Lokesh kumawat
March 5, 2025Done
Abhilasha
March 7, 2025Okay sir
VIKRAM GAHLOT
March 8, 2025👍
Diksha Chhipa
August 20, 2025Done
Khushi Mehra
August 20, 2025Stripping and re probing of the gel during western blotting give clear and sequential detection with different antibodies
Varsha Tatla
August 20, 2025Not cleared
Neelam Sharma
August 20, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Aman Choudhary
August 21, 2025Option D is correct
stripping and reprobing during Western blotting give sequential detection of different antibodies instead of running the multiple gels
Manisha
August 21, 2025Done ✅
Aafreen Khan
August 21, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting
Bcoz it saves time and resources and allows sequential detection
Sakshi yadav
August 21, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Avni
August 21, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Bhawna Choudhary
August 21, 2025Correct answer is D stripping and reprobing
Santosh Saini
August 21, 2025Stripping and reprobing of gel after transferring to a nitrocellulose membrane during western blotting
Sakshi Kanwar
August 22, 2025D … AS stripping leads to removal of Ab after a blot and reprobe will provide antibody to another antigen and we can detect bands within the blot
Soniya Shekhawat
August 22, 2025Stripping is remove the all antibody after blot and repropping are provided antibody on the another antigen them we detect band on blot.
karishma
August 22, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Parul Yadav
August 22, 2025Striping and reprobing Western blots is the most effective method to differentiate co-migrating proteins.
Arushi
August 22, 2025Stripping and reprobing
Priya dhakad
August 23, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to nitrocellulose membrane while doing Western blotting.
Bharti Yadav
August 23, 2025D. Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Devika
August 24, 2025Answer d. Stripping and reprobing
Kanishka Parnami
August 24, 2025Stripping and reprobing
Sneha Kumawat
August 24, 2025Stripping and reprobing of gel during western blotting
Yogita
August 24, 2025Stripping reprobing of the gel after transferring to a nitrocellulose membrane while doing western blotting
Aakanksha Sharma
August 24, 2025By stripping and reprobing
Himanshi
August 24, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
AKANKSHA RAJPUT
August 24, 2025option d is correct
Shivani
August 24, 2025Subjecting the technique of stripping or reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting is the correct answer .
Bhavana kankhedia
August 24, 2025Subjecting the technique of stripping/ reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Neeraj Sharma
August 24, 2025Option D is correct as we can strip the first probe after detection of one protein and then again reprobe it via another probe to detect the other protein presence
Anisha Beniwal
August 24, 2025Option -D
Ayush Dubey
August 24, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting
Tanvi Panwar
August 24, 2025Using striping and reprobing techniques.
Priyanka Verma
August 24, 2025option 4 is correct – using stripping and reprobing techniques
Sonam Saini
August 24, 2025Opetion D is right answer
Surendra Doodi
August 24, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting
Falguni Prakash
August 24, 2025D
Kajal
August 25, 2025Option D is right
Priyanka Meena
August 26, 2025Subjective the technique of stripping/reprobing of the gel after transferring to nitrocellulose members while doing Western blotting
Neha Yadav
August 27, 2025Stripping and reprobing of the gel
Manisha choudhary
August 27, 2025Stripping and reprobing
Kanica Sunwalka
August 28, 2025stripping and reprobing
Shriyanshi verma
August 30, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Asha Gurzzar
August 30, 2025D will be correct stripping and reprobing
Alec
August 30, 2025option d – subjecting the technique of stripping the gel after transferring to nitrocellulose membrane while doing western blotting.
Pratibha Jain
August 30, 2025Correct answer is option (D)
Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Kirti agarwal
August 30, 2025Stripping and probing on membrane
anjani sharma
August 30, 2025Stripping,reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Anisha jakhar
August 31, 2025Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.
Shobha Kanwar
August 31, 2025Correct answer is option (D)
Subjecting the technique of stripping/reprobing of the gel after transferring to a nitrocellulose membrane while doing Western blotting.