HEK 293T cells were lysed 16 hours post-transfection with 200 μL of M-PER lysis buffer (Thermo Scientific) plus protease inhibitors. After 10 min on a rotary shaker at room temperature, lysates were collected and centrifuged for 15 min (94 rcf; 4°C). The supernatants were collected and combined with 6X Laemmli sample buffer, followed by heating for 10 min at 65˚C. The resulting samples were subjected to electrophoresis on a 10% SDS-PAGE gel and then transferred onto PVDF membranes (20 V, overnight, at 4 °C). Membranes were then blocked for 1 h with 5% BSA in TBS with 1% Tween (TBST), followed by incubation with primary antibody (Anti-mCherry antibody (Cell Signaling); 1:1000 dilution in 5% BSA – TBST; Anti-GAPDH antibody (ThermoScientific); 1:1000 dilution in 5% BSA – TBST) overnight at 4°C on a platform rocker. The membranes were then washed 3 × 5 min each with TBST and incubated with the appropriate secondary antibody in 5% BSA – TBST for 2 hours at room temp. After washing 3 × 5 min with TBST, the membranes were exposed to a chemiluminescent substrate for 5 min and imaged using an Azure imaging station.
Anti mcherry antibody
Manufactured by Cell Signaling Technology
The Anti-mCherry Antibody is a reagent used for the detection and visualization of mCherry, a red fluorescent protein commonly used as a reporter in biological research. This antibody specifically binds to mCherry, allowing for the localization and quantification of mCherry-tagged proteins or cells.
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Lab products found in correlation
3 protocols using anti mcherry antibody
1
Western Blot Analysis of Transfected HEK 293T Cells
2
Western Blot Analysis of mCherry in HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Transfected HEK293T cells were lysed with 200 μl of M-PER lysis buffer (Thermo Fisher Scientific) containing 1× Halt protease–phosphatase inhibitor cocktail (Thermo Fisher Scientific). After 10 min on ice, lysates were collected and centrifuged for 15 min (94 rcf; 4 °C). The supernatants were combined with Laemmli SDS sample buffer (Alfa Aesar) and incubated at 65 °C for 10 min. The resulting lysates were subjected to electrophoresis on a 10% SDS-PAGE gel and then transferred onto polyvinylidene fluoride membranes (20 V, 800 min). Membranes were then blocked for 1 h with 5% bovine serum albumin (BSA) in 1× Tris-buffered saline (TBS) with 1% Tween (TBST), followed by incubation with primary antibody (anti-mCherry antibody [Cell Signaling] 1:1000 dilution in 5% BSA–TBST; anti-GAPDH antibody [Invitrogen] 1:1000 dilution in 5% BSA–TBST) overnight at 4 °C on a platform rocker. The membranes were then washed 3× for 5 min each with TBST and incubated with the appropriate secondary antibody in 5% BSA–TBST (1 h; room temperature). After washing 3× for 5 min with TBST, the membranes were exposed to a chemiluminescent substrate for 5 min and imaged with an Azure cSeries imaging station.
3
Western Blot Analysis of Transfected HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected HEK293T cells were lysed with 200 µL of M-PER lysis buffer (Thermo Scientific) containing 1X Halt protease-phosphatase inhibitor cocktail (Thermo Scientific). After 10 min on a rotary shaker at room temperature, lysates were collected and centrifuged for 15 min (94 rcf; 4˚C). The supernatants were combined with Laemmli SDS sample buffer (Alfa Aesar) and incubated at 65˚C for 10 min. The resulting lysates were subjected to electrophoresis on a 10% SDS-PAGE gel and then transferred onto PVDF membranes (20 V, 800 min). Membranes were then blocked for 1 h with 5% BSA in 1x TBS with 1% Tween (TBST), followed by incubation with primary antibody (Anti-mCherry antibody (Cell Signaling) 1:1000 dilution in 5% BSA -TBST; Anti-GAPDH antibody (Invitrogen) 1:1000 dilution in 5% BSA -TBST) overnight at 4°C on a platform rocker. The membranes were then washed 3x for 5 min each with TBST and incubated with the appropriate secondary antibody in 5% BSA -TBST (1 h; room temperature). After washing 3x for 5 min with TBST, the membranes were exposed to a chemiluminescent substrate for 5 min and imaged with an Azure cSeries imaging station.
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