1. Protein Extraction from Paraffin Embedded Sections:
Composition of Protein Extraction Buffer
Conditions for Protein Extraction
1) 200µL RIPA buffer + 2% SDS (PI and PhospI)
Ikeda, et al
100oC for 20 min (on heating block)
60oC for 2hr (water bath)
Tissue lysates then centrifuged at 15,000xg for 20 min at 4oC
Supernatants collected at -20 oC
2) 200µL
20mM Tris-HCl, pH 7.0
2% SDS
200mM DTT
Shi, et al
3) 200µL
6M Guanidine HCl
100mM Tris HCl, pH 7.0
Jiang, et al J of Proteome Research 2007
Incubate in the lysis buffer for 4oC (in cold room on rocking platform)
4) 200µL
Addis, et al Proteomics 2009
80oC for 2hr (with Thermomixer agitation 750rpm)
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
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 10µm thick were used per protein extraction condition
5 conditions were used for protein extraction

2. First Blot: 5/24/11
Determining the best protocol for protein extraction from FFPE (formalin fixed paraffin embedded sections)
Lanes 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Short Exposure
Long Exposure
Lane 1: Marker
Lane 2: Shed Cells (FOP sample) ~50µg
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

3. First Blot Stripped and Reprobed: 5/25/11
Determining the best protocol for protein extraction from FFPE (formalin fixed paraffin embedded sections)
Lanes 1 2 3 4 5 6 7 1 2 3 4 5 6 7
Long Exposure
Short Exposure
Lane 1: Marker
Lane 2: Shed Cells (FOP sample) ~50µg
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

4. FFPE sample Protein Extraction
Blot 1: 6/2/11
FFPE sample Protein Extraction
Lanes 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Mrker
Lane 1: Shed Cells (FOP sample) ~13µg
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) ~25µg
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)

5. ***Motorcycle man sample
p-SMAD
HBO9-7 Lysate
HBO9-9 Lysate
HBO9-19 Lysate
HBO9-20 Lysate
Normal Shed Cell Lysate
Polymorphisms
c270 C>T
c delT
c A>a/g
c G>g/a
c642+7 C>c/t
c690 G>A
1131 C>c/t
c G>g/a
c G>g/a
138 after stop G>A
Samples
HB09-7
HB09-9
HB08-19
HB08-20***
***Motorcycle man sample

7. Lanes 1 2 3 4 5 6 7 8
Mrker
Lane 1: Shed Cells (Norm sample) ~20µg
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