Western blots were conducted using the following primary antibodies: rabbit anti-AC3 (ab14778, Abcam, Cambridge, MA, USA), rabbit anti-AC7 (ab14782, Abcam, Cambridge, MA, USA), and rabbit anti-AC9 (ab191423, Abcam, Cambridge, MA, USA). The secondary antibodies used were: goat anti-rabbit HRP labeled (1:60,000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA); and goat anti-mouse HRP labeled (1:60,000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Proteins of interest were detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and Fusion Fx7 imager (MBI Lab Equipment, Kirkland, QC, Canada). Quantification of immunoblots was performed by densitometry using ImageJ software (from Wayne Rasband, National Institute of Health (NIH), USA).
Ab191423
Manufactured by Abcam
Sourced in United States
Ab191423 is a primary antibody product available from Abcam. It is a monoclonal antibody that recognizes a specific target protein. This product is suitable for use in common laboratory techniques, such as Western blotting and immunohistochemistry. Further details about the target, specificity, or intended applications are not provided in order to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab10983, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pmsf protease inhibitor, by Merck Group (2 mentions)
Goat anti mouse hrp, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Las1000 plus chemiluminescence imaging system, by Fujifilm (1 mentions)
Dab kit, by Agilent Technologies (1 mentions)
Sa00004 2, by Proteintech (1 mentions)
H8070, by Solarbio (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Axiocam icc1 camera, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti filaggrin, by Abcam (1 mentions)
Rabbit anti involucrin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
Ab61778, by Abcam (1 mentions)
5 protocols using ab191423
1
Quantification of Protein Expression Levels
2
Quantitative Analysis of Metabolic Regulators
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Tissues and cells were lysed in RIPA buffer containing 1 mmol/L PMSF protease inhibitor (P7626, Sigma). The protein concentrations were then measured using a BCA Protein Assay kit (Thermo Fisher Scientific, 23227). Primary antibodies against UCP1 (#ab10983, Abcam, 1:2000), AC9 (ab191423, Abcam, 1:1000), phospho-AMPKα (Thr172, #2535, CST, 1:1000), total-AMPKα (#5831, CST, 1:1000), phospho-HSL (Ser563, #4139, CST, 1:1000), HSL (#4107, CST, 1:1000), CPT1α (#12252, CST, 1:1000), MEK5 (#40737, CST, 1:1000), PPARγ (#2443, CST, 1:1000), and FASN (#3189, CST, 1:1000) were used according to the manufacturer’s instructions. Primary antibodies were incubated in a blocking buffer at 4 °C overnight. Secondary Alexa antibodies from Life Technologies were then added for 1 h. Detection was performed by chemiluminescence (ImmobilonTM Western, Millipore Corporation, Billerica, MA, USA), captured using a FUJI LAS 1000-plus chemiluminescence imaging system. The protein density was quantified and analyzed using Image J 1.52a software.
3
Protein Quantification and Western Blot Analysis
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Tissues and cells were lysed in RIPA buffer containing 1 mmol/L PMSF protease inhibitor (P7626, Sigma). The protein concentrations were then measured using a BCA Protein Assay kit (Thermo Fisher Scientific, 23227). Primary antibodies against phospho-STAT3 (Tyr705, #9131S, CST, 1:1000), STAT3 (#12640S, CST, 1:1000), UCP1 (#ab10983, Abcam, 1:2000), β-Tubulin (#2146S, CST, 1:1000) and AC9 (ab191423, Abcam, 1:1000) were used according to the manufacturer’s instructions. Primary antibodies were incubated in blocking buffer at 4 °C overnight. Secondary Alexa antibodies from Life Technologies were then added for 1 h. The protein density was quantified and analyzed using Image J software.
4
Immunohistochemical Analysis of ADCY9 Expression
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Formalin was used to treat tumor tissue sections (4 µm thick), followed by bedding the sections in paraffin. Xylene anhydrous ethanol was used to remove paraffin while catalase was blocked by 3% hydrogen peroxide. The sections were incubated with ADCY9 antibodies (1:1,000, ab191423, Abcam, UK) for 24 h at 4 ℃, then sections were incubated with secondary antibodies (1:1,000, SA00004-2, Proteintech) for 2 h at 37 ℃. The nuclei were stained with hematoxylin (H8070, Solarbio, China) after DAB kit (K5007, DAKO, Denmark) staining. Image J was used to calculate the integrated density of the DAB staining positive area to estimate the expression of ADCY9.
5
Immunofluorescence Staining of Skin Tissue
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For immunofluorescence staining, tissues were embedded in Tissue-Tek O.C.T Compound (Sakura Finetek, CA, USA) and 6 µm thick sections were fixed in acetone at −20 °C before staining. The following antibodies were used and incubated in a dark room at room temperature for 45 min: rabbit anti-involucrin (Abcam, Cambridge, MA, USA), rabbit anti-filaggrin (Abcam, Cambridge, MA, USA), rabbit anti-keratin 10 (Abcam, Cambridge, MA, USA), mouse anti-Ki67 IgG1 (BD Biosciences, CA, USA), rabbit anti-AC9 (ab191423, Abcam, Cambridge, MA, USA) and rabbit anti-beta 2 adrenergic receptor (ab61778, Abcam, Cambridge, MA, USA). Tissues were then incubated with Alexa Fluor 488 donkey anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG (1:1600, Thermofisher Scientific, CA, USA) for 30 min also at room temperature. Nuclear counter staining using DAPI (SouthernBiotech, AL, USA) was then effected on different samples. Each tissue was observed using a Zeiss Axio Imager M2 microscope with an AxioCam ICc1 camera. The quantification of immunofluorescence staining was performed by densitometry using ImageJ software (from Wayne Rasband, National Institute of Health (NIH), USA).
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