Antibody for staining PI4P was purchased from Echelon Biosciences. Antibody against PV 3D was produced in Cameron lab. Mouse monoclonal anti-GAPDH antibody (10R-G109a) was purchased from Fitzgerald Industries. All secondary antibodies used for immunofluorescence were purchased from Invitrogen. Secondary antibodies for western blotting were purchased from Amersham GE Healthcare (rabbit anti-HRP) and Cell Signaling (mouse anti-HRP). Rabbit polyclonal anti-eIF2D antibody (12840-1-AP) and anti-eIF2A antibody (11233-1-AP) were purchased from Proteintech. Rabbit anti-phospho (S51)-eIF2α (9721) and rabbit anti-eIF2α (9722) were purchased from Cell Signaling Technology. Rabbit anti-phospho (T446)-PKR (32036) and rabbit anti-PKR (32506) were purchased from Abcam.
Anti eif2d antibody 12840 1 ap
Manufactured by Proteintech
The Anti-eIF2D antibody (catalog number 12840-1-AP) is a primary antibody that recognizes the eIF2D protein. eIF2D is a translation initiation factor involved in the regulation of protein synthesis. This antibody can be used to detect and study the expression of eIF2D in various experimental systems.
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3 protocols using anti eif2d antibody 12840 1 ap
1
Immunostaining and Western Blotting
2
Quantification of Protein Silencing Efficiency
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The silencing efficiency was quantified by western blots using rabbit polyclonal anti-eIF2D antibody (12840–1-AP) from Proteintech and mouse polyclonal anti-GAPDH antibody (sc-1377179) from Santa Cruz Biotechnology. For that, 20 μg of protein extracts were loaded on 10% polyacrylamide SDS-PAGE. After migration, proteins were transferred to an Immobilin-P membrane (Millipore) at 10 V for 1 hr in a semi-dry apparatus (Trans-Blot SD) on a PVDF (polyvinylidene fluoride) membrane that had been previously activated with 100% methanol for few seconds and a transfer buffer pH 8 (25 mM Tris; 200 mM glycine; 20% ethanol). After transfer, the membrane was saturated for 2 hr by blocking buffer (5% milk, 0.05% Tween-20, PBS 1×). Primary antibodies were added at dilutions recommended by the manufacturers in blocking buffer, the membranes were incubated overnight at 4°C. Then, the membranes were washed three times by PBST (PBS 1×; 0.05% Tween-20) to remove the excess of primary antibodies. Then, membranes were incubated with secondary HRP-conjugated antibody for 1 hr at room temperature followed by three washing steps. The signal produced by reaction between HRP and ECL (Kit ECL Plus Western Blotting Detection System, GE Healthcare) was detected by chemiluminescence using imaging Chemidoc (Biorad).
3
Quantification of Protein Silencing Efficiency
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The silencing efficiency was quantified by western blots using rabbit polyclonal anti-eIF2D antibody (12840-1-AP) from Proteintech, mouse monoclonal anti-eIF4E antibody (sc-9976) and mouse polyclonal anti-GAPDH antibody (sc-1377179) from Santa Cruz Biotechnology.
For that, 20 µg of protein extracts were loaded on 10% polyacrylamide SDS PAGE. After migration, proteins were transferred to an Immobilin-P membrane (Millipore®) at 10 Volts for 1 h in a semi-dry apparatus (Trans-Blot® SD) on a PVDF membrane (PolyVinyliDene Fluoride) that had been previously activated with 100% methanol for few seconds and a transfer buffer pH 8 (25 mM Tris; 200 mM glycine; 20% ethanol). After transfer, the membrane was saturated for 2h by blocking buffer (5% milk, 0.05% Tween-20, PBS 1X).
Primary antibodies were added at dilutions recommended by the manufacturers in blocking buffer, the membranes were incubated overnight at 4°C. Then, the membranes were washed three times by PBST (PBS 1X; 0.05% Tween-20) to remove the excess of primary antibodies.
Then, membranes were incubated with secondary HRP-conjugated antibody for 1h at room temperature followed by three washing steps. The signal produced by reaction between HRP and ECL (Kit ECL Plus Western Blotting Detection System, GE Healthcare®) was detected by chemiluminescence using imaging Chemidoc (Biorad®).
For that, 20 µg of protein extracts were loaded on 10% polyacrylamide SDS PAGE. After migration, proteins were transferred to an Immobilin-P membrane (Millipore®) at 10 Volts for 1 h in a semi-dry apparatus (Trans-Blot® SD) on a PVDF membrane (PolyVinyliDene Fluoride) that had been previously activated with 100% methanol for few seconds and a transfer buffer pH 8 (25 mM Tris; 200 mM glycine; 20% ethanol). After transfer, the membrane was saturated for 2h by blocking buffer (5% milk, 0.05% Tween-20, PBS 1X).
Primary antibodies were added at dilutions recommended by the manufacturers in blocking buffer, the membranes were incubated overnight at 4°C. Then, the membranes were washed three times by PBST (PBS 1X; 0.05% Tween-20) to remove the excess of primary antibodies.
Then, membranes were incubated with secondary HRP-conjugated antibody for 1h at room temperature followed by three washing steps. The signal produced by reaction between HRP and ECL (Kit ECL Plus Western Blotting Detection System, GE Healthcare®) was detected by chemiluminescence using imaging Chemidoc (Biorad®).
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