StainFree & Normalisation. Eric Niedo. Mercredi 17 Juin June 18, 2015
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1 StainFree & Normalisation Eric Niedo June 18, 2015 Mercredi 17 Juin 2015
2 Protein Workflow 2
3 Le V3!!! La normalisation 5 Séparation des protéines: Visualisation de la migration: Transfer des Proteines: Vérification du transfert: Validation du Western Blot : TGX Stain-Free Precast Gels enable rapid separation of proteins TGX Stain-Free Precast Gels and the Chemi Doc MP imager enable immediate visualization of protein separation The Trans-Blot Turbo system enables rapid and efficient transfer of proteins across a wide range of molecular weights The Chemi Doc MP paired with Stain-Free technology enables instant verification of protein transfer Chemiluminescence and multiplex fluorescent Western blot detection with validation of quantitation using Stain- Free total protein normalization
4 Gels TGX StainFree 4 TGX (Tris Glycine extended) Precast Gels Mini-PROTEAN TGX Precast Gels Criterion TGX Precast Gels 10 cm Criterion Gel 13.3 x 8.7 cm 5 cm Mini-PROTEAN Gel 8.6 x 6.7 cm 0 cm 0 cm 5 cm 10 cm 15 cm
5 Gels TGX StainFree 5 Long shelf life 12 months 7.5%, 10%, 12%, Any kd, 4-15%, 4-20%, 8-16% Precast Gel Other long shelf life (Bis-Tris) Recommended Times (User Manuals) Volts Minutes Running Buffer 200* 50 MOPS / SDS 200* V 60 TGX 200V 28 Laemmli TGX 300V 20 Laemmli TGX 400V 10 Laemmli MES / SDS (Retards protein migration) Tris-Acetate (Large protein separation) Run time = 12min
6 Le StainFree Visualisation de la migration 5 TGX Stain-Free Precast Gels & ChemiDoc Touch System pour une Visualisation immédiate de la séparation des protéines Stain Free Coomassie R D Comparison of Criterion Stain Free vs R-250 Coomassie of total protein samples 1) Precision Plus Standard, 2) mouse liver lysate, 3) rat liver, 4) mouse thymus, 5) rat thymus, 6) HeLa, 7) E. coli lysate, 8) ProteoMiner treated human serum, 9) untreated human serum Samples were run on a 12+2 well 4-20% Tris-HCl Criterion Stain Free gel. The gel was imaged on the Stain Free system and then stained with R-250 Coomassie, followed by imaging on a Bio-Rad GS-800 densitometer. The visible bands in the complex protein samples are comparable
7 Le Transfert Tank Blotting with Tris-HCl gels 90 à 120 min transfer times with hand-cast gels 2 mini / 2 midi gels per run 10+ minutes setup time 90 Min Tank Blotting with TGX gels 60 min transfer times with hand-cast gels 2 mini / 2 midi gels per run 10+ minutes setup time 60 Min Trans-Blot Turbo 3 à 10 min transfer times with TGX gels 2 mini gels per run Simplified consumable packs easy setup 3-10 Min
8 Le Stain Free Vérifier le transfert 5 Le ChemiDoc Touch System associé à la technologie StainFree pour une Vérification immédiate du transfert des protéines Stain-Free Gel after electrophoresis Stain-Free gel after transfer Stain-Free blot after transfer AnykD 4-20% AnykD 4-20% AnykD 4-20% Trans-Blot Turbo (3 min) AnykD 4-20% AnykD 4-20% AnykD 4-20% Trans-Blot Turbo (10 min) Precision Plus Unstained standard (outer lanes) and E.coli (inner lanes) by Mini PROTEAN TGX Stain-Free AnykD or 4-20% gradient gel and imaged with ChemiDoc MP. Proteins were blotted to PVDF membrane with Trans-Blot Turbo system for 3 minutes (Turbo protocol) or 10 min (High MW protocol). Gel was re-imaged with ChemiDoc MP after transfer to check transfer efficiency. The transferred blot was imaged with ChemiDoc MP to confirm unbiased transfer.
9 Le Stain Free Valider le Western Blot 5 Le StainFree à la meilleure gamme dynamique pour une normalisation optimum Tubulin Immunodetection: µg Ponceau S: µg Sypro Ruby: µg Stain Free: µg
10 Le V3!!! Valider le Western Blot 5 La normalisation grâce aux protéines de ménage est ce la meilleure approche? Some journals ask for multiple normalization We were asked by the reviewer for 5 independent validation experiments for our WB data Setting up/running western blots with multiple analytes is very time consuming or simply not feasible Total protein normalization is more stable and less prone to experimental conditions
11 Le V3!!! Valider le Western Blot La normalisation grâce aux protéines totales est plus solide!!! A B A: LCL lysate (30 µg total protein per lane) of untreated (o) and irradiated (*) samples separated by Criterion SF TGX AnykD gel and blotted to PVDF membrane. Stain-Free signal was imaged with ChemiDoc MP. B: Immunofluorescence imaging of DNA replication factor MCM7 and housekeeping protein GAPDH with ChemiDoc MP at different wavelengths. Monoclonal antibodies against MCM7 (mouse) and GAPDH (rabbit) were diluted 1:1,000 and 1:2,500 respectively. Secondary antibodies from Rockland were antirabbit DyLight 549 (1:10,000) and antimouse DyLight 649 (1:20,000). Quantities for each lane: MCM7 signal (target protein) GAPDH signal for HKP normalization SF signal for total protein normalisation MCM7 (un-normalized) Normalization within each lane: MCM7/GAPDH for HKP normalization MCM7/SF for total protein normalisation = MCM7 Regulation: Ø irradiated samples Ø controls
12 MCM 7 Regulation Factor Le V3!!! Valider le Western Blot La normalisation grâce aux protéines totales est plus solide!!! 1 0,9 0,8 0,7 0,6 Expected Factor 0,5 0,4 0,3 0,2 0, ± 35% p < ± 24% p < ± 29% p < MCM 7 (Raw Data) MCM 7 (SF Norm.) MCM 7 (GAPDH Norm.) Statistical analysis reveals good correlation of all data with expected regulation factor but Stain-Free normalization shows lowest variation of data and best p-value indicating perfect statistical significance for this normalization approach.
13 Le V3!!! 13 Valider le Western Blot
14 Littérature
15 Merci de votre attention 15
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