Lysates were prepared in RIPA buffer (Cell Signaling Technologies) with 1mM PMSF per manufacturer's instructions. Proteins were separated on a NuPAGE gel (Invitrogen) and transferred to a PVDF membrane using the XCell II blotting system (Invitrogen). Membranes were blocked with 5% nonfat milk and incubated with primary antibody overnight at 4°C. Primary antibodies were purchased from Cell Signaling Technologies: IRAK1 (#4504), phospho-IkBa (#2859), IkBa (#4814), phospho-p38 MAPK (#4511), p38 MAPK (#8690), phospho-p44/42 MAPK (ERK1/2, #4370), p44/p42 MAPK (ERK1/2, #4695), NFkB2 p100/p52 (#4882), phospho-NFkB p65 (#3033), NFkB p65 (#8242) phospho-TBK1 (#5483), TBK1 (#3504). CXCL1 (PA1-29220, Thermo Fisher Scientific), GAPDH (CB1001), and total actin antibody (MAB1501) were purchased from EMD Millipore. HuR (sc-5261), IRAK1 (sc-5288), phospho-MAPKAPK2 (sc-293139), MAPKAPK2 (sc-393609), and TTP (sc-374305) were purchased from Santa Cruz Biotechnology. TRAF2 (592) was purchased from Medical & Biological Laboratories. Following washing, membranes were incubated with HRP-tagged anti-mouse IgG (1706516, Bio-Rad) or anti-rabbit IgG (NA934, GE Healthcare) and developed using SuperSignal West Pico or Femto substrate (Thermo Fisher Scientific).
Gapdh cb1001
Manufactured by Merck Group
GAPDH (CB1001) is a laboratory reagent produced by Merck Group. It is an enzyme that catalyzes the sixth step of glycolysis, the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. This reaction is critical for the production of energy in the form of ATP during cellular respiration.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Phospho nf kb p65, by Cell Signaling Technology (1 mentions)
Nf kb p65, by Cell Signaling Technology (1 mentions)
P44 p42 mapk, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Phospho ikba, by Cell Signaling Technology (1 mentions)
Nf kb2 p100 p52, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
Irak1, by Cell Signaling Technology (1 mentions)
Phospho tbk1, by Cell Signaling Technology (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Super signal chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Sds page gel, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
4 protocols using gapdh cb1001
1
Immunoblotting Protocol for Inflammatory Signaling
2
Western Blot Analysis of Cell Cycle Regulators
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Cell culture medium RPMI 1640 and DMEM:F12 were from Gibco. Primary antibodies for western blots were obtained from Sigma for SAMHD1 (HPA047072), Cell Signaling for Cyclin E1 (4129S) and Cyclin A2 (4656S), Abcam for Histone H3 phosphoS10 (ab14955), and EMD Millipore for GAPDH (CB1001). Secondary HRP-conjugated antibodies against rabbit (7074S) and mouse (7076S) were from Cell Signaling Technology. Nitrocellulose membranes were from Bio-Rad and Super Signal chemiluminescence reagent was from Thermo Scientific. DCP-Bio1 was from Xoder.
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from the cells using RIPA lysis buffer with presence of protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined using the Pierce BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein (20 μg) were loaded and resolved on 10% SDS-PAGE gels (Thermo Fisher Scientific). After transfer onto nitrocellulose membranes, samples were probed with primary antibodies for TMEM49/VMP1 (D1Y3E, 1:1,000; Cell Signaling, Danvers, MA), GAPDH (CB1001, 1:1,000; Millipore, Danvers, MA). Secondary antibodies were ECL anti-rabbit immunoglobulin G (IgG) (Sigma-Aldrich) and ECL anti-mouse IgG (Thermo Fisher Scientific) (1:5,000) corresponding to the primary antibody.
4
Western Blot Analysis of Chikungunya Viral Proteins
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Cells were collected by trypsinization and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors. Samples were run on Tris-glycine SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon) for Western blot detection. Blots were exposed with chemiluminescent regents and imaged with a charge-coupled device (CCD)-based detection system (Amersham). The following antibodies were used: DDX56 (A302-979A, RRID:AB_10750607 ; Bethyl), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; CB1001, RRID:AB_2107426 ; Millipore), lamin B1 (ab16048, RRID:AB_443298 ; Abcam), tubulin (T6199, RRID:AB_477583 ; Sigma-Aldrich), CHIKV nsP1 (11–13020; Abgenex), CHIKV nsP2 (10–10024; Abgenex), and CHIKV nsP3 (11–13022; Abgenex).
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