Immunoblotting for Apoptosis Markers

Immunoblotting was performed as previously described [8 (link)]. For pro-caspase 3, cleaved caspase 3, pro-caspase 9, BiP and CHOP detection, cell lysates were resolved on 10% or 12% SDS/PAGE. After blocking with 5% non-fat milk in TBS/Tween-20, membranes were incubated with pro-caspase 3, cleaved caspase 3, pro-caspase 9, BiP or CHOP (1 : 1000, all), antibodies at 4 °C overnight. For ubiquitinated protein detection, cell lysates were resolved on a 4–20% linear gradient polyacrylamide gel (BioRad, Hercules, CA, USA) before detection with poly-ubiquitinated conjugates antibody (1 : 1000). Blots were washed and the appropriate secondary antibodies applied. Protein signal was visualized with ECL reagents (Pierce). Blots were then stripped and reprobed using GAPDH antibody as a loading control.

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Liu H., Chen J., Li W., Rose M.E., Shinde S.N., Balasubramani M., Uechi G.T., Mutus B., Graham S.H, & Hickey R.W. (2015). Protein disulfide isomerase as a novel target for cyclopentenone prostaglandins: implications for hypoxic ischemic injury. The FEBS journal, 282(10), 2045-2059.

Publication 2015

4–20% linear gradient polyacrylamide gel

Antibodies Antibody Caspase 3 Cell Chop Gapdh Membranes Milk Poly Polyacrylamide gel Pro caspase 3 Pro caspase 9 Protein Sds page Tween 20

Corresponding Organization : Children's Hospital of Pittsburgh

Other organizations : VA Pittsburgh Healthcare System, Geriatric Research Education and Clinical Center, University of Windsor

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Variable analysis

independent variables

Antibody concentrations (1:1000 for pro-caspase 3, cleaved caspase 3, pro-caspase 9, BiP, CHOP, and poly-ubiquitinated conjugates)
Antibody incubation time (overnight at 4 °C)

dependent variables

Levels of pro-caspase 3, cleaved caspase 3, pro-caspase 9, BiP, CHOP, and poly-ubiquitinated proteins

control variables

SDS/PAGE gel percentage (10% or 12% for pro-caspase 3, cleaved caspase 3, pro-caspase 9, BiP, CHOP; 4-20% linear gradient for ubiquitinated proteins)
Blocking solution (5% non-fat milk in TBS/Tween-20)
Washing and secondary antibody application
Protein signal visualization (ECL reagents)
Reprobing with GAPDH antibody as a loading control

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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