Grafts and SPCs were lysed in fresh extraction buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with a protease inhibitor and phosphatase inhibitor (Gold Biotechnology, St. Louis, MO, USA). The extracted proteins were separated on a 15% SDS-polyacrylamide gel and electroblotted onto a polyvinylidene fluoride membrane. The membrane was blocked using 5% skim milk-containing TBST for 60 min at 20–25°C and incubated with primary antibodies against Bcl-2 (Affinity), Bax (Affinity), cytochrome c (Affinity), cleaved-caspase-3(Affinity), cleaved-PARP (Affinity), and PCNA (Abcam) overnight at 4℃. The membranes were then washed with TBST and incubated with goat anti-mouse IgG (R&D Systems, Inc., Minneapolis, MN, USA) and goat anti-rabbit IgG (R&D Systems) secondary antibodies. Bound antibodies were detected using an electrochemiluminescence detection system (Amersham Life Science, Arlington Heights, IL, USA). β-actin was used as the loading control.
Protease inhibitor and phosphatase inhibitor
Manufactured by GoldBio
Sourced in United States
Protease inhibitor and phosphatase inhibitor are laboratory reagents used to prevent the degradation of proteins and dephosphorylation of phosphoproteins, respectively, during sample preparation and analysis. They are commonly used in cell and tissue lysis buffers to maintain the integrity of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Fresh extraction buffer, by Merck Group (2 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Goat anti mouse igg, by R&D Systems (1 mentions)
Electrochemiluminescence detection system, by Cytiva (1 mentions)
Cleaved parp, by Affinity Biosciences (1 mentions)
Cytochrome c, by Affinity Biosciences (1 mentions)
Cleaved caspase 3, by Affinity Biosciences (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
Af5002, by Affinity Biosciences (1 mentions)
Af2006, by Affinity Biosciences (1 mentions)
Af4001, by Affinity Biosciences (1 mentions)
Haf007, by R&D Systems (1 mentions)
Af6456, by Affinity Biosciences (1 mentions)
Af2002, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Haf008, by R&D Systems (1 mentions)
2 protocols using protease inhibitor and phosphatase inhibitor
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Splenocytes were lysed in fresh extraction buffer (Sigma-Aldrich, St. Louis, MO,
USA) supplemented with a protease inhibitor and phosphatase inhibitor (Gold
Biotechnology, St. Louis, MO, USA). The extracted proteins (20 μg) were
separated on a 15% SDS-polyacrylamide gel and electroblotted onto a
polyvinylidene fluoride membrane. The membrane was blocked using 5% milk
dissolved in TBST for 60 min at 20–25°C and incubated overnight at 4°C with
primary antibodies against p-p38 (AF4001, Affinity Biosciences, Changzhou,
China), p38 (AF6456, Affinity Biosciences) p-p65 (AF2006, Affinity Biosciences),
p65 (AF5006, Affinity Biosciences), p-IκBα (AF2002, Affinity Biosciences) and
IκBα (AF5002, Affinity Biosciences). The membranes were then washed with TBST
and incubated with goat anti-mouse IgG (1:1000; HAF007, R&D Systems, Inc.,
Minneapolis, MN, USA) and goat anti-rabbit IgG (1:1000; HAF008, R&D Systems,
Inc., Minneapolis, MN, USA). Bound antibodies were detected using an
electrochemiluminescence detection system (Amersham Life Science, Arlington
Heights, IL, USA). β-Actin was used as the control and to ensure equal protein
loading.
USA) supplemented with a protease inhibitor and phosphatase inhibitor (Gold
Biotechnology, St. Louis, MO, USA). The extracted proteins (20 μg) were
separated on a 15% SDS-polyacrylamide gel and electroblotted onto a
polyvinylidene fluoride membrane. The membrane was blocked using 5% milk
dissolved in TBST for 60 min at 20–25°C and incubated overnight at 4°C with
primary antibodies against p-p38 (AF4001, Affinity Biosciences, Changzhou,
China), p38 (AF6456, Affinity Biosciences) p-p65 (AF2006, Affinity Biosciences),
p65 (AF5006, Affinity Biosciences), p-IκBα (AF2002, Affinity Biosciences) and
IκBα (AF5002, Affinity Biosciences). The membranes were then washed with TBST
and incubated with goat anti-mouse IgG (1:1000; HAF007, R&D Systems, Inc.,
Minneapolis, MN, USA) and goat anti-rabbit IgG (1:1000; HAF008, R&D Systems,
Inc., Minneapolis, MN, USA). Bound antibodies were detected using an
electrochemiluminescence detection system (Amersham Life Science, Arlington
Heights, IL, USA). β-Actin was used as the control and to ensure equal protein
loading.
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