SDS–PAGE workflow in protein biochemistry — What is the correct initial sample treatment before electrophoretic separation on a polyacrylamide gel containing the anionic detergent SDS?

Difficulty: Easy

Treat with a reducing agent (e.g., β-mercaptoethanol or DTT) and SDS (anionic detergent), then load for electrophoresis
First run electrophoresis, then add an oxidizing agent, followed by SDS afterward
Treat with an oxidizing agent, then add SDS, then run electrophoresis
No pretreatment is used; proteins are run in their native state by default
Dialyze proteins against water only, then perform native PAGE without SDS

Correct Answer: Treat with a reducing agent (e.g., β-mercaptoethanol or DTT) and SDS (anionic detergent), then load for electrophoresis

Explanation:

Introduction:
SDS–PAGE is a foundational technique to separate proteins primarily by molecular mass. The preparative chemistry before loading is critical: proteins are denatured and, if required, disulfide bonds are reduced to ensure subunits migrate according to size rather than shape or native charge.

Given Data / Assumptions:

SDS is an anionic detergent that binds proteins roughly in proportion to length.
Reducing agents such as β-mercaptoethanol or DTT cleave disulfide bonds.
Electrophoresis is performed in a polyacrylamide matrix under an electric field.

Concept / Approach:
SDS unfolds proteins and confers a near-constant negative charge-to-mass ratio. Reducing agents remove disulfide constraints so each polypeptide chain migrates independently. Correct sample preparation precedes electrophoresis to standardize migration behavior across diverse proteins.

Step-by-Step Solution:

1) Mix sample with SDS-containing sample buffer to denature and coat polypeptides.2) Add a reducing agent (β-mercaptoethanol or DTT) to break disulfide bonds; heating often assists denaturation.3) Load the treated sample into wells and apply voltage to separate mainly by molecular mass.

Verification / Alternative check:
When reduction is omitted, covalently linked subunits co-migrate as larger species. Including reducing agent yields band patterns that match subunit molecular masses expected from known sequences.

Why Other Options Are Wrong:

b) Treatment after electrophoresis is illogical; separation must follow denaturation.c) Oxidizing agents create or preserve disulfides, counter to SDS–PAGE goals.d) Native conditions do not reflect SDS–PAGE methodology.e) Dialysis and native PAGE would separate by charge/shape, not SDS mass-based separation.

Common Pitfalls:
Confusing native PAGE with SDS–PAGE; neglecting reduction when analyzing multimeric proteins; underheating samples leading to incomplete denaturation.

Final Answer:
Treat with a reducing agent and SDS, then perform electrophoresis.

Discussion & Comments

No comments yet. Be the first to comment!

SDS–PAGE workflow in protein biochemistry — What is the correct initial sample treatment before electrophoretic separation on a polyacrylamide gel containing the anionic detergent SDS?

More Questions from Gel Electrophoresis

Discussion & Comments