Slide 1

Protein Extraction from Paraffin Embedded Sections: Used Protocol from Qiagen for: Deparaffinization of FFPE Samples Cut Directly from a Block 5 serial sections cut at 10m thick were used per protein extraction condition 5 conditions were used for protein extraction Composition of Protein Extraction BufferConditions for Protein Extraction 1) 200L RIPA buffer + 2% SDS (PI and PhospI) Ikeda, et al 100 o C for 20 min (on heating block) 60 o C for 2hr (water bath) Tissue lysates then centrifuged at 15,000xg for 20 min at 4 o C Supernatants collected at stored @ -20 o C 2) 200L 20mM Tris-HCl, pH 7.0 2% SDS 200mM DTT Shi, et al 100 o C for 20 min (on heating block) 60 o C for 2hr (water bath) Tissue lysates then centrifuged at 15,000xg for 20 min at 4 o C Supernatants collected at stored @ -20 o C 3) 200L 6M Guanidine HCl 100mM Tris HCl, pH 7.0 Jiang, et al J of Proteome Research 2007 Incubate in the lysis buffer for 72hr @ 4 o C (in cold room on rocking platform) 4) 200L 20mM Tris-HCl, pH 7.0 2% SDS 200mM DTT Addis, et al Proteomics 2009 100 o C for 20 min (on heating block) 80 o C for 2hr (with Thermomixer agitation 750rpm) Tissue lysates then centrifuged at 15,000xg for 20 min at 4 o C Supernatants collected at stored @ -20 o C 5) 1mL Qiazol (0.77g of tissue, so I rounded to 1mL of Qiazol) Protocol: Lysis of Fatty Tissue Using the TissueRuptor User-Developed Protocol: Isolation of the protein fraction from the organic phase of QIAzol Reagent-treated fatty tissue samples Slide 2 First Blot: 5/24/11 Determining the best protocol for protein extraction from FFPE (formalin fixed paraffin embedded sections) Short Exposure Long Exposure Lanes 132456798101112 132456798101112 Lane 1: Marker Lane 2: Shed Cells (FOP sample) ~50g Lane 3: Extraction Method 1 Lane 4: Extraction Method 2 Lane 5: Extraction Method 3 Lane 6: Extraction Method 4 Lane 7: Extraction Method 5 Loaded by volume, protein estimation was not performed Slide 3 Short Exposure Lanes 1324567 1324567 Long Exposure Lane 1: Marker Lane 2: Shed Cells (FOP sample) ~50g Lane 3: Extraction Method 1 Lane 4: Extraction Method 2 Lane 5: Extraction Method 3 Lane 6: Extraction Method 4 Lane 7: Extraction Method 5 Loaded by volume, protein estimation was not performed First Blot Stripped and Reprobed: 5/25/11 Determining the best protocol for protein extraction from FFPE (formalin fixed paraffin embedded sections) Slide 4 Blot 1: 6/2/11 FFPE sample Protein Extraction Lanes 13 2 4 5 6 7 8 13245678 Mrker Lane 1: Shed Cells (FOP sample) ~13g Lane 2: X Lane 3: HB09-6 Lane 4: HB09-7 Lane 5: HB09-9 Lane 6: HB08-19 Lane 7: HB09-20 Lane 8: HB09-20 (6/1/2011) Mrker Lane 1: Shed Cells (FOP sample) ~25g Lane 2: X Lane 3: HB09-1 Lane 4: HB09-8 Lane 5: HB09-9 Lane 6: HB09-11 Lane 7: HB09-20 Lane 8: HB09-20 (6/1/2011) Slide 5 Normal Shed Cell Lysate HBO9-7 Lysate HBO9-9 Lysate HBO9-19 Lysate HBO9-20 Lysate p-SMAD Polymorphisms c270 C>T c67-236 delT c67-236 A>a/g c.330+60 G>g/a c642+7 C>c/tc690 G>A1131 C>c/t c1395+45 G>g/a c1396-93 G>g/a 138 after stop G>A Samples HB09-7 HB09-9 HB08-19 HB08-20*** ***Motorcycle man sample Slide 6 Slide 7 Lanes 1 3 2 4 5 6 7 8 Mrker Lane 1: Shed Cells (Norm sample) ~20g Lane 2: HB09-10 Norm Lane 3: HB09-1 Lane 4: HB09-7 Lane 5: HB09-8 Lane 6: HB09-11 Lane 7: HB08-19 Lane 8: HB09-20