Sequence of Steps in Western Blotting: Understanding the Process
Sequence of steps in western blotting is
P. Protein denaturation
Q. Hydrogen peroxide reduction
R. Primary antibody binging
S. Transfer onto membrane
Correct Sequence of Steps in Western Blotting
Western blotting is a widely used protein detection technique in molecular biology and biochemistry. It allows researchers to separate, transfer, and identify specific proteins from complex mixtures. The correct sequence of steps in Western blotting is:
- (P) Protein Denaturation
- (S) Transfer onto Membrane
- (R) Primary Antibody Binding
- (Q) Hydrogen Peroxide Reduction
Thus, the correct order is: P → S → R → Q.
Step-by-Step Explanation of Western Blotting
1. Protein Denaturation (P)
- Purpose: To unfold proteins into a linear form for accurate separation.
- Process:
- The protein sample is mixed with SDS (Sodium Dodecyl Sulfate), which coats proteins with a negative charge.
- Heating the sample further denatures the proteins by breaking non-covalent bonds.
2. Transfer onto Membrane (S)
- Purpose: To transfer separated proteins from the gel to a membrane (usually nitrocellulose or PVDF).
- Process:
- Proteins are transferred electrophoretically, using an electric field to move them onto the membrane.
- The membrane immobilizes the proteins for further analysis.
3. Primary Antibody Binding (R)
- Purpose: To detect specific proteins using an antibody that binds to the target protein.
- Process:
- The membrane is blocked using non-fat dry milk or BSA (Bovine Serum Albumin) to prevent non-specific binding.
- The primary antibody (specific to the target protein) is added and incubated.
4. Hydrogen Peroxide Reduction (Q)
- Purpose: To detect the bound antibody through a chemiluminescent or colorimetric reaction.
- Process:
- A secondary antibody conjugated with horseradish peroxidase (HRP) or alkaline phosphatase (AP) is introduced.
- Hydrogen peroxide is added as a substrate for HRP, producing a detectable signal (chemiluminescence or color change).
Why Is Western Blotting Important?
Western blotting is used for:
✅ Disease diagnosis (e.g., HIV detection, cancer biomarker identification)
✅ Protein expression studies
✅ Post-translational modification analysis
✅ Drug development and biomedical research
Conclusion
The Western blotting process follows a specific sequence: Protein denaturation → Transfer onto membrane → Primary antibody binding → Hydrogen peroxide reduction. This method is crucial in protein analysis, medical research, and diagnostics, offering high specificity and sensitivity for detecting target proteins.
60 Comments
Ujjwal
March 8, 2025✔️✔️
Suman bhakar
March 8, 2025Done
Pooja jat
March 10, 2025Done.
Arushi
April 9, 2025👍✔️
Asha Gurzzar
August 20, 2025P-s-r-q
Neelam Sharma
August 20, 2025(P) Protein Denaturation
(S) Transfer onto Membrane
(R) Primary Antibody Binding
(Q) Hydrogen Peroxide Reduction
P-S-R-Q
Nilofar Khan
August 20, 2025Correct order p-s-r-q
Khushi Mehra
August 20, 2025Protein denaturation, transfer on membrane, 1° antibody binding, hydrogen peroxide reduction
Correct answer is P-S-R-Q
Kavita choudhary
August 20, 2025Protin denaturation , transfer in to membrane, primary antibody binding ,hydrogen peroxide reduction
Deepika Sheoran
August 20, 2025Option d is correct
Protein denaturation
Transfer on to membrane
Primary Antibody binding
Hydrogen peroxide.
Mohd juber Ali
August 21, 2025Option d is correct
Denature – transfer on membrane – 1° ab binding – H peroxide reduction
Manisha
August 21, 2025Done ✅
Aman Choudhary
August 21, 2025Option D is correct
Denaturation ➡️ Membrane Transfer ➡️ Primary Ab ➡️ Hydrogen peroxide
Priyanka choudhary
August 22, 2025Right answer is option d
the order is P → S → R → Q
Khushi Vaishnav
August 21, 2025Protein Denaturation
Transfer onto Membrane
Primary Ab Binding
Hydrogen Peroxide Reduction
Payal Gaur
August 21, 2025P-protein denaturation
S-protein transfer on membrane
R- 1° antibody binding
Q- hydrogen peroxide reduction
Sakshi yadav
August 21, 2025(P) Protein denaturation
(S) Transfer onto membrane
(R) Primary antibody binding
(Q) Hydrogen peroxide reduction
Meera Gurjar
August 21, 2025Psrq is right
Aafreen Khan
August 21, 2025P-S-R-Q
Sheikh Aleen
August 21, 2025P S R Q
Avni
August 21, 2025Protein denaturation,Transfer onto membrane ,Primary antibody binding ,Hydrogen peroxide reduction.
PSRQ
Divya rani
August 21, 2025Protein denaturation
Transfer on membrane
1° ab binding
Hydrogen peroxide reduction
PSRQ
Santosh Saini
August 21, 2025Protein denaturation, transfer on membrane, pri. Antibody binding, hydrogen peroxide reduction
Ankita Pareek
August 21, 2025Denaturation of protein
Transfer onto nitrocellulose membrane
Antibody binding
Hydrogen peroxide reduction
Simran Saini
August 21, 2025P, S, R, Q.
Protein denaturation.
Transfer onto membrane.
Primary antibody binding.
Hydrogen peroxide reduction.
shruti sharma
August 22, 2025p-s-r-q
Priyanka choudhary
August 22, 2025The right answer is D
the order is: P → S → R → Q.
karishma
August 22, 2025PSRQ
Soniya Shekhawat
August 23, 2025Steps of Western blotting is protein denaturation them transfer on membrane them primary antibody binding final step hydrogen peroxide reducing.
Kirti agarwal
August 23, 2025Psrq
Priya dhakad
August 23, 2025(P) Protein Denaturation
(S) Transfer onto Membrane
(R) Primary Antibody Binding
(Q) Hydrogen Peroxide Reduction
So the correct option is P-S-R-Q
Priya khandal
August 23, 2025Psrq
Niti Tanwar
August 23, 2025Psrq
Sakshi Kanwar
August 23, 2025Firstly protein is denatured then is trasfered to membrane ..primary antibody binds then hydrogen peroxide is used for observation ….option D
Kajal
August 23, 2025Initially protein is denatured then transferred to membrane after that primary antibody treatment n after that all we use hydrogen peroxide reduction.
Bharti Yadav
August 23, 20251.Protein denaturation
2.Transfer to membrane
3.Primary antibody binding
4.Hydrogen peroxide reduction
Yogita
August 24, 2025Psrq
Sneha Kumawat
August 24, 2025Protein denaturation
Transfer on to membrane
Primary antibody binding
Hydrogen peroxide reduction
Mitali saini
August 24, 2025The correct sequence of steps in Western blotting is:
(P) Protein Denaturation
(S) Transfer onto Membrane
(R) Primary Antibody Binding
(Q) Hydrogen Peroxide Reduction
Thus, the correct order is: P → S → R → Q.
Shivani
August 24, 2025Steps in Western blotting are- (P) Protein Denaturation
(S) Transfer onto Membrane
(R) Primary Antibody Binding
(Q) Hydrogen Peroxide Reduction
So option(D). – P-S-R-Q is correct .
Neeraj Sharma
August 24, 2025Hydrogen peroxide helps to detect the binded antibody hence option D is correct
Tanvi Panwar
August 24, 2025The correct sequence is – protein denaturation, transfer to membrane,primary antibody binding and hydrogen peroxide reduction.
Surendra Doodi
August 24, 2025Protein Denaturation -Transfer onto Membrane- Primary Antibody Binding-Hydrogen Peroxide Reduction
Sonam Saini
August 24, 2025Right sequence psrq
Priyanka Verma
August 24, 2025the correct order is – P S R Q
Falguni Prakash
August 24, 2025D option
Swati Choudhary
August 24, 2025(P) Protein Denaturation
(S) Transfer onto Membrane
(R) Primary Antibody Binding
(Q) Hydrogen Peroxide Reduction
Kashish yadav
August 25, 2025PSRQ
Bhavana kankhedia
August 25, 2025the order is PSRQ
Kajal
August 25, 2025Protein denaturion
Transfer on to membrane
Primary antibody binding
Hydrogen peroxide reduction
Priyanka Meena
August 26, 2025P. Protin denaturation
S. Tranfer of member
R. Primary antibody bind
Q. Hydrogen peroxide reductive
Muskan Yadav
August 26, 2025(P) Protein Denaturation
(S) Transfer onto Membrane
(R) Primary Antibody Binding
(Q) Hydrogen Peroxide Reduction
The correct answer is – P,S,R,Q
Neha Yadav
August 27, 2025P- S-R-Q
Manisha choudhary
August 27, 2025P-s-r-q
First protein denaturantion
Second transfer on membrane
Third primary antibody
Third H2O2 DENGE
Kanica Sunwalka
August 28, 2025done
Shriyanshi verma
August 30, 2025Protin denaturation , transfer in to membrane, primary antibody binding ,hydrogen peroxide reduction
Alec
August 30, 2025The correct sequence is P – S – R – Q
Pratibha Jain
August 30, 2025the correct order is P → S → R → Q.
Anisha jakhar
August 31, 2025P-S-R-Q
Shobha Kanwar
August 31, 2025Protin denaturation , transfer in to membrane, primary antibody binding ,hydrogen peroxide reduction