Cell pellets from selected iPSC clones were lysed in RIPA buffer (Cell Signaling Technologies) and proteins were electrophoretically separated on Nu-PAGE polyacrylamide gels (Invitrogen). Proteins were transferred to nitrocellulose membranes (Invitrogen), blocked in 3% milk in TBS-Tween, and probed with antibodies to detect TPM1 (1:1000, Cell Signaling Technologies) or β-Actin (1:5000, Sigma). Secondary antibodies were anti-Rabbit IgG-HRP or anti-Mouse IgG-HRP (Bio-Rad). Western blots were imaged using a Chemi-Doc Imaging System (Bio-Rad).
Nu page polyacrylamide gels
Manufactured by Thermo Fisher Scientific
Nu-PAGE polyacrylamide gels are precast electrophoresis gels used for the separation and analysis of proteins. They are designed to provide consistent and reproducible results in the separation and visualization of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg hrp, by Bio-Rad (3 mentions)
Anti mouse igg hrp, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Cyclin e2, by Abcam (1 mentions)
Non immune igg, by Santa Cruz Biotechnology (1 mentions)
6 protocols using nu page polyacrylamide gels
1
Western Blot Analysis of iPSC Protein Expression
2
Western Blot Analysis of iPSC Proteins
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Cell pellets from selected iPSC clones were lysed in RIPA buffer (Cell Signaling Technologies) and proteins electrophoretically separated on Nu-PAGE polyacrylamide gels (Invitrogen). Proteins were transferred to nitrocellulose membranes (Invitrogen), blocked in 3% milk in TBS-Tween, and probed with antibodies to detect TPM1 (1:1000, Cell Signaling Technologies) or β-Actin (1:5000, Sigma). Secondary antibodies were anti-Rabbit IgG-HRP or anti-Mouse IgG-HRP (Bio-Rad). Western blots were imaged using a Chemi-Doc Imaging System (Bio-Rad).
3
Protein Separation and Quantification
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Proteins were separated on 4–12% Bis–Tris–HCl (pH 7.0) NuPAGE polyacrylamide gels (Invitrogen) and stained with G-colloidal Coomassie Brilliant Blue. Lanes of the Coomassie-stained gel were cut into 23 slices and processed for mass spectrometry as previously described (42 (link)). A comparison of the number of peptides identified for each protein in two samples was used as a reliable indication of the relative number of proteins when they are analyzed using identical chromatographic and mass-spectrometric conditions and the same mass spectrometer.
4
Western Blot Analysis of iPSC Protein Expression
5
Comprehensive Cell Lysate and Fractionation Protocol
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Collection of whole cell lysates [20 ], chromatin [21 (link)] and sucrose gradient fractions of centrosomes [22 (link)] were performed as described. Lysates were separated using NuPage polyacrylamide gels (Invitrogen) prior to transfer to PVDF membranes. Western blotting, immunofluorescence and PLA antibodies are: Cdc6 (180.2), CDK2 (M2, D12), centrin-2 (S-19), cyclin E1 (HE12), estrogen receptor α (HC20), NPAT (C-19, 27), SAP145 (A-20), γ-tubulin (C-11) (Santa Cruz Biotechnology); cyclin E2 (Epitomics); p21 (610234) and p27 (610242) (BD Biosciences); αGST [23 (link)]. Immunoprecipitation antibodies are: CDK2 (C-19), cyclin E1 (C-19), NPAT (C-19, 27), non-immune IgG (Santa Cruz Biotechnology), and cyclin E2 (Epitomics). Specificity of cyclin E1 and E2 antibodies was demonstrated in [15 (link),19 (link)]. Additionally, we show specific loss of cyclin E1 and cyclin E2 immunofluorescence signal with siRNA treatment to cyclin E1 (Additional file 4 ) and cyclin E2 (Figure 3 ).
6
Quantitative Protein Analysis via Gel Electrophoresis
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Proteins were separated on 4-12% Bis-Tris-HCl (pH 7.0) NuPAGE polyacrylamide gels (Invitrogen) and stained with G-colloidal Coomassie Brilliant Blue. Entire lanes of the Coomassie-stained gel was cut into 23 slices and processed for mass spectrometry as previously described (39) . A comparison of the number of peptides identified for each protein in two samples is a reliable indication of the relative amount of proteins when they are analyzed using identical chromatographic and mass-spectrometric conditions and the same mass spectrometer.
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