SDS-PAGE & Western Blotting

Step one induce cells with IPTG

Activates the lack promoter → drives over expression of GFP fusion protein

2. Life cells with BPER containing DNaseI and lysosome

Break open cell, DNase reduce reduces viscosity by degrading DNA, license zone digest, bacterial cell walls

3. Boil samples in buffer with SDS and BME

SDS denatures proteins, and gives uniform negative charge, BME breaks dice sulfide bonds, boiling, fully denatures proteins

4. Run samples on SDS-PAGE

Separates proteins by size do to SDS normalization of charge

5. Transfer proteins to nitrocellulose membrane

Moves proteins from gel to stable membrane for antibody detection

6. Stain membrane with Ponceau

Confirms successful transfer and equal protein loading across lanes

7. Incubate membrane with milk solution (blocking)

Blocked nonspecific binding sites to prevent antibody background

8. Probe with primary mouse anti-GFP antibody

Provide specificity by binding the target GFP-containing protein

9. Probe with secondary goat anti-mouse HRP-conjugated antibody

Find the primary antibody and amplify signal via HRP enzyme

10. Incubate with HRP colorimetric substrate

HRP convert substrate into visible signal → allows protein detection

SDS (sodium lauryl sulfate) does

Denatures proteins, coats proteins in a uniform negative charge

Why does SDS matter in SDS-PAGE

Without SDS protein’s migrate based on charge and shape

With SDS proteins migrate only by molecular weight

What is PAGE

Polyacrylamide Gel Electrophoresis → gel separates denatured proteins by molecular weight