The following reagents and materials were used: anti-ALDH1B1 (ref. sc-374090) was purchased from Santa Cruz; anti-PI16 (ref. PAS 31882) from Thermo; anti-P70 S6K beta (ref: ab184551) from Abcam; and anti-Bcl-xL (ref. 2764), anti-pAkt (Ser473) (ref. 4060), anti-Akt (ref. 4685), anti-pMEK1/2 (Ser217/221) (ref. 9154), anti-MEK1/2 (ref. 9126), anti-pERK1/2 (Thr202/Tyr204) (ref. 4370), anti-ERK1/2 (ref. 9102), anti-pPKA (Thr197) (ref. 5661), anti-PKA (ref. 4782), anti-pSEK1/MKK4 (Ser257/Thr261) (ref. 9156), anti-SEK1/MKK4 (ref. 9152), anti pSAPK/JNK (Thr183/Tyr185) (ref. 9255), anti pSAPK/JNK (ref. 9252S), anti pMKK3-6 (Thr183/Tyr185) (ref. 9231), anti MKK3 (ref. 5674), anti p-p38 MAPK (Thr180/Tyr182) (ref. 4511), anti p38 MAPK (ref. 9212) were purchased from Cell Signaling. Electrophoresis reagents were purchased from Biorad and trypsin from Promega.
Anti pka
Manufactured by Abcam
Sourced in United States
Anti-PKA is a primary antibody that specifically binds to and detects the regulatory subunit of Protein Kinase A (PKA). PKA is a key enzyme involved in signal transduction pathways and cellular processes. The Anti-PKA antibody can be used to study the expression and localization of the PKA regulatory subunit in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti creb, by Abcam (3 mentions)
Anti ppka, by Abcam (2 mentions)
Anti p erk1 2, by Abcam (1 mentions)
Anti p p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
Anti psapk jnk, by Abcam (1 mentions)
Anti erk1 2, by Abcam (1 mentions)
Electrophoresis reagents, by Bio-Rad (1 mentions)
Anti bcl xl, by Abcam (1 mentions)
Anti pmek1 2, by Abcam (1 mentions)
Anti mek1 2, by Abcam (1 mentions)
Anti mkk3, by Cell Signaling Technology (1 mentions)
Anti p38 mapk, by Cell Signaling Technology (1 mentions)
Trypsin, by Promega (1 mentions)
Anti akt, by Abcam (1 mentions)
Col 1, by Bio-Rad (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Gapdh, by Merck Group (1 mentions)
7 protocols using anti pka
1
Quantitative Immunoblotting for Signaling Proteins
2
Osteogenic Protein Expression in MC3T3-E1 Cells
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The treated MC3T3-E1 cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentrations were quantified by BCA Protein Assay reagent (Pierce; Thermo Fisher Scientific, Inc.). Total protein (20 µg) was run on 12% sodium dodecyl sulfate-polyacrylamide gel and electro-transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.), which were then blocked with 5% non-fat dry milk in Tris-buffered saline, followed by the incubation with the primary antibodies anti-collagen type I (Col-I; 1:5,000; 130 kDa; cat. no. ab34710), anti-ALP (1:500; 39 kDa, cat. no. ab83259), anti-osteopontin (OPN; 1:1,000; 66 kDa, cat. no. ab8448), anti-Runx2 (1:1,000; 57 kDa; cat. no. ab23981), and anti-BMP-2 (1:1,000; 45 kDa; cat. no. ab14933; all from Abcam), anti-PKA (1:1,000; 42 kDa; cat. no. 4782), anti-p-cAMP response element-binding protein (CREB; 1:1,000; 43 kDa; cat. no. 9198) and anti-CREB (1:1,000; 43 kDa; cat. no. 9197; all from Cell Signaling Technology (CST)) overnight at 4°C. Following the primary antibodies, the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibodies (cat. no. ab205718; Abcam) at 4°C for 1 h. Protein bands were detected with an enhanced chemiluminescence detection system (EMD Millipore), and GAPDH (1:1,000; 36 kDa; cat. no. ab8245; Abcam) was used as the internal control.
3
Gastric Cancer Cell Line Cultivation
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Human gastric cancer cell lines, including BGC-823, MGC, SGC-790, HGC, and MKN-45 were purchased from the American Type Culture Collection in 2015. All cells were cultured in DMEM, supplemented with 10% fetal bovine serum, and were grown in a humidified atmosphere containing a 95% air, 5% CO2 mixture at 37°C. Anti-AM, anti-P-JNK, anti-JNK, anti-P-PKA, anti-PKA, and anti-LC3-II/I antibodies were obtained from Abcam Technology (Cambridge, MA). Anti-cleaved-caspase3, anti-caspase3, anti-P-AKT, anti-AKT, anti-Beclin, and anti-CRLR antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GAPDH, anti-Bax, anti-Bcl2, anti-RAMP1, anti-RAMP2, and anti-RAMP3 antibodies were obtained from Cell Signaling Technology (Beverly, MA).
4
Protein Expression Analysis by SDS-PAGE and Western Blotting
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SDS–PAGE (10% and 4% gels) and Western blotting analysis were used in the present study. The immunoreactive bands were visualized by ECL solution, and the images were quantified by a UVP gel imager. The primary antibodies used were as follows: anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) (Abcam, USA, mouse, 1/100,00), anti-CREB (phospho S133) (Abcam, USA, rabbit, 1/5,000); anti-CREB (Abcam, USA, rabbit, 1/500), anti-BDNF (Abcam, USA, mouse, 0.2–2 μg/mL); anti-β catenin (Abcam, USA, rabbit, 1/5,000), anti-GSK3β (Abcam, USA, mouse, 1/500); anti-GSK3β (phospho Y216) (Abcam, USA, rabbit, 1/500), and anti-PKA (Abcam, USA, rabbit, 1/500).
5
Glomerular Protein Expression Analysis
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Glomerular protein was extracted with SDS protein lysate (KeyGEN, Nanjing, China), separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. PVDF membrane was blocked with 5% skim milk containing 0.05% Tween-20 and incubated with individual primary antibodies, including anti-FN (Santa Cruz Biotechnology, Dallas, TX), anti-RAGE, anti-PKC (Sigma-Aldrich, Shanghai, China), anti-PKA (Abcam, UK), anti-iNOS (Abcam, UK), anti-SOD1 (Santa Cruz, USA) and anti-actin (Sigma-Aldrich, Shanghai, China). Then HRP-conjugate secondary antibody was utilized and color development achieved with enhanced chemiluminescence (ECL) reagent (EMD Millipore, Billerica, MA, USA).
6
Cocaine-Induced CREB Phosphorylation
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Cocaine, D1R antagonist [R(+)-SCH-23390 hydrochloride] and D2R antagonist [S(-)-raclopride(+)-tartrate salt] were purchased from the Sigma Chemical Company (St. Louis, MO, USA). All chemicals were dissolved immediately in physiological saline before use. The CART and GAPDH primers, M-MLV reverse transcriptase and AccuPower PCR PreMix were purchased from Bioneer Co. (Seoul, Korea). The 2X SYBR Green Master Mix was purchased from Applied Biosystems (Carlsbad, CA, USA). The anti-CART and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phospho[Ser-133] CREB, anti-CREB, and anti-PKA antibodies were purchased from Abcam (Cambridge, MA, USA). The Vectastain ABC reagent was purchased from Vector Laboratories Co. (Burlingame, CA, USA). The Cyclic AMP XP™ Assay Kit was purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Protein G Sepharose 4 Fast Flow was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). The PKA Assay kit was purchased from Millipore Co. (Bedford, MA, USA). The [γ-32P] ATP was purchased from Perkin Elmer, Inc. (Covina, CA, USA). All other materials were of the highest grade available and were obtained from normal commercial sources.
7
Hippocampal Protein Expression Analysis
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The total proteins of hippocampal tissues and primary neurons were isolated using radioimmunoprecipitation assay buffer (RIPA). The protein concentration of all tissue lysates was determined with a bicinchoninic acid assay (BCA) protein assay kit. Same amount of total protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, Unite States). The membranes were blocked with 5% non-fat powdered milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature (RT) and then incubated overnight at 4°C with the appropriate primary antibodies: anti-Arc, anti-PKA (phospho T197; p-PKA), anti-ERK1 + ERK2 (phospho T202 + T204; p-ERK1/2), and anti-CREB (phospho S133; p-CREB) (Abcam, Cambridge, MA, Unite States). After washing in TBST, the membrane was incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, and protein bands were visualized using the Immun-StarTM HRP Chemiluminescence Kit (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal loading control.
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