IEMR

1. IEMR Proteins: Separation, purification and quantification Per Kristian Lunde Institute for experimental medical research IEMR, Oslo Norway www.iemr.no

2. IEMR • Main objectivesofthislecture: • Separationof proteins by gel electrophoresis • Detectionof proteins in gels and membranes • Isolationof proteins from different cellular compartments by centrifugation IEMR, Oslo Norway www.iemr.no

3. IEMR • Proteins: • Chains of aminoacids (AA, ≈20 different) • More than 50 AA in a chain is usuallytermed proteins, less than 50 AA = peptides (Titin: more than 34000AA => Mw > 3800 kDa) • Different types of proteins: Structural, membrane, soluble….. • A protein mayconsistofone or several AA chains Ryanodine receptor, Efremov et al, Nature 2015, 517, 39 IEMR, Oslo Norway www.iemr.no

4. IEMR • Protein structure are often devided in: • Primary structure: AA-sequence • Secondary structure: three-dimensional structure of the AA-chain (alfahelix and betasheet) • Tertiary structure: folding of the AA-chain (hydrofobic interactions, hydrogen bindings, Van der Waals forces, salt bridges) • Quarternary structure: Protein complex consisting of several peptide chains (subunits) giving a functional protein IEMR, Oslo Norway www.iemr.no

5. IEMR Separation of proteins: - Electrophoresis • Sentrifugation/sedimentation • Precipitation IEMR, Oslo Norway www.iemr.no

6. IEMR Protein gel electroforesis: Sample preparation: Samplebuffer which contain among other SDS (sodium dodecyl sulphate) which binds to the protein and gives it a negative charged surface. In addition it breaks hydrogen- and disulfide bonds, and by that straighten out the protein. IEMR, Oslo Norway www.iemr.no

7. IEMR Protein gelektroforese: A polyacrylamide gel (PAGE) consist of polacrylamide which is crosslinked with bisacrylamid. Dependent of the polyacrylamide concentration we will get a gel with different size of the pores in the gel. The polyacrylamide gel is moulded between to glasplates with a ”stacking” gel on top, and a ”separating” gel beneath. The samples are applied in wells in the stacking gel. Due to the negative charged surface of the protein (due to the SDS), the proteins will move in the gel when a electric field is put over the gel. In the stacking gel the proteins are concentrated to a sharp band, while they will be separated dependent of size in the separating gel. IEMR, Oslo Norway www.iemr.no

8. IEMR 250 150 200 75 50 37 MHC 25 20 15 15 % SDS-PAGE 4 % SDS-PAGE/Glycerol kDa IEMR, Oslo Norway www.iemr.no

9. IEMR Protein gel electroforesis Gel Staining Coomassie Silver Sypro ruby ProQ diamond …. Blotting Antibody binding Detection IEMR, Oslo Norway www.iemr.no

10. IEMR Western blotting: - Method used for detection of a specific protein in a sample containing several proteins. - Based on antigen-antibody binding - Semi quantitativ IEMR, Oslo Norway www.iemr.no

11. IEMR Western blotting: Blotting: Transfer of the proteins in a gel to a membrane, nitrocellulose or PVDF, which has a high affinity for proteins. Blocking: Blocking of non-spesific binding by saturate the area of the membrane that has not bound the proteins from the gel transfer, by incubating the membrane in a solution of protein, e.g. BSA or dry milk IEMR, Oslo Norway www.iemr.no

12. IEMR Antibodies : Monoclonal: Binds to one epitope on a protein For example, antibodies which binds to a site with a phophorylated aminoacid Polyclonal: Binds to several epitops on a protein IEMR, Oslo Norway www.iemr.no

13. IEMR Antibody binding and detection: Primary antibody: A specific antibody for your target protein Secondary antibody: A spesies specific antibody, often linked to a reporter enzyme, such as AP or HRP. Detection: • Horseradish peroxidase (HRP) • Alkaline phophatase (AP) -Cy3/Cy5 (Fluorescence) -Immonogold -Autoradiography IEMR, Oslo Norway www.iemr.no

14. IEMR Western blot on tissue from controls and mice with knockout of SERCA2 in skeletal muscle IEMR, Oslo Norway www.iemr.no

15. IEMR Protein gel electroforesis Gel Staining Coomassie Silver Sypro ruby ProQ diamond …. Blotting Antibody binding Detection IEMR, Oslo Norway www.iemr.no

16. Detection of MLC1 and 2 Soleus EDL 250 MHC 150 200 75 50 Actin Tropomyosin Troponin T 37 25 MLC1s MLC1f 20 MLC2s 15 MLC2f KTR EX KTR EX KTR EX KTR EX CHF SHAM CHF SHAM Sypro Ruby: Stains all proteins

17. Phosphorylation of MLC1 and 2 Soleus EDL 250 MHC 150 200 75 50 Actin Tropomyosin Troponin T 37 25 MLC1s MLC1f 20 MLC2s 15 MLC2f KTR EX KTR EX KTR EX KTR EX CHF SHAM CHF SHAM ProQ Diamond: Stains phosphorylated proteins

18. IEMR Separationof proteins: • Homogenisation • Sentrifugation • Alternating resuspending and sentriguging in different buffers • Total homogenat • Sentrifuge => Cytoplasmic (soluble) proteins in supernatant • Resuspend/sentrifuge => Nuclear proteins in supernatant • Resuspend/sentrifuge => Membrane proteins in supernatant • Resuspend/sentrifuge => Cytoskeletal proteins in supernatant => rest in pellet IEMR, Oslo Norway www.iemr.no

19. IEMR Total Cyt Nuc Mem C.S. Pellet Std Std 250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa K.H.Hortemo 10% SDS-PAGE, Coomassie stained IEMR, Oslo Norway www.iemr.no

20. IEMR Total Cyt Nuc Mem C.S. Pellet Std Std 250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa K.H.Hortemo 10% SDS-PAGE, O-GlcNac IEMR, Oslo Norway www.iemr.no

21. IEMR Total Cyt Nuc Mem C.S. Pellet Std Vinculin SDH Serca2 Histone H3 GAPDH K.H.Hortemo IEMR, Oslo Norway www.iemr.no

22. IEMR IL18 antibody A Soleus EDL IL18=24kDa 15 kDa 20 kDa 25 kDa 37 kDa 50 kDa 75 kDa 100 kDa IEMR, Oslo Norway www.iemr.no

23. IEMR IL18=24kDa IL18 antibody B IL18 antibody C Soleus EDL Soleus EDL 20 kDa 10 kDa 15 kDa 25 kDa 100 kDa 50 kDa 75 kDa 37 kDa IEMR, Oslo Norway www.iemr.no

24. IEMR IEMR, Oslo Norway www.iemr.no