A researcher needs to identify if Protein A is localized in the ER in a human cell line. The cell has an RFP-tagged protein that marks the ER in this cell. Following are the proposed experiments:

A. Express Protein A fused to GFP at the N-terminus in the cells, followed by microscopy to check for colocalization with RFP.
B. Express Protein A fused to GFP at the C-terminus in the cells, followed by microscopy to check for colocalization with RFP.
C. Immunofluorescence staining of A, followed by microscopy to check for colocalization with RFP.
D. Isolating the ER by differential centrifugation and checking for co-purification of Protein A with RFP.

Which one of the following options represents experiments that would most likely identify localization of Protein A in the ER?

Correct option is B

The best approach to confirm ER localization combines microscopy-based colocalization with biochemical fractionation.

• Immunofluorescence (C) is a classical and reliable method for detecting protein localization via antibodies and microscopy.

• Differential centrifugation (D) isolates cellular organelles like the ER, and checking co-purification provides biochemical evidence for localization.

• Fusion proteins tagged at different termini (A and B) can affect the protein's targeting or function, so both are often tested, but this may sometimes give ambiguous results if the tag disrupts targeting signals.

Therefore, the combination B, C and D (Option 2) provides a comprehensive and reliable approach:

• B: Tagging at C-terminus, possibly preserving the targeting signal better than N-terminus.

• C: Immunofluorescence confirms localization visually without relying on fusion proteins.

• D: Biochemical validation with ER isolation and co-purification.

Important Key Points:

• Colocalization microscopy helps visualize if two proteins occupy the same cellular compartment.

• Fusion tags (GFP/RFP) allow live-cell imaging but may alter protein targeting depending on tag placement.

• Immunofluorescence staining uses antibodies specific to the protein, providing direct localization evidence.

• Differential centrifugation separates cell components by density, allowing biochemical verification of localization.