Anti ldha

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

Anti-LDHA is a laboratory product designed to detect and quantify the expression of the LDHA (Lactate Dehydrogenase A) protein in biological samples. It is a specific antibody that can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression and localization of LDHA in cells and tissues.

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Anti-LDH A (LDHA) (2 citations) Anti-LDHA antibody (2 citations)

13 protocols using anti ldha

Purification of Murine Brain Mitochondria

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The brains of Adult C57BL6/J mice were dissected, and mitochondria were purified as described.^{43 (link)} Briefly, brains were extracted in ice-cold isolation buffer (sucrose 250 mM, Tris 10 mM, EDTA 1 mM, NaF 2 mM, pH7.6) and homogenized with a Teflon potter. Homogenates were centrifuged at 1500 g for 5 min (4 °C). The supernatant was then centrifuged at 12 500 g (4 °C). The pellet was collected, and both centrifugations were repeated. To purify mitochondria, the final pellet was resuspended in 400 μl of isolation buffer, layered on top of a discontinuous Ficoll gradient (10 and 7% fractions), and centrifuged at 100 000 g for 1 h (4 °C). The following antibodies were used as purification controls: anti-SDHA (1 : 20 000, Abcam, Cambridge, UK) and anti-TOM20 (1 : 1000, Santa Cruz Biotechnology) as mitochondrial markers; anti-LDHa (1 : 500, Santa Cruz Biotechnology) as cytosolic marker; anti-tubulin (1 : 1000, Sigma-Aldrich) as cytoskelton marker; and anti-synaptophysin (1 : 20 000, Millipore, Schwalbach, Germany) as synaptosome marker.

Serrat R., Mirra S., Figueiro-Silva J., Navas-Pérez E., Quevedo M., López-Doménech G., Podlesniy P., Ulloa F., Garcia-Fernàndez J., Trullas R, & Soriano E. (2014). The Armc10/SVH gene: genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation. Cell Death & Disease, 5(4), e1163-.

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Quantifying Protein Expression Profiles

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Western blot analysis was performed as given by Shang et al. [33 (link)]. The antibodies were applied as follows: anti-LDHA (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP2 (1:1,000; Cell Signaling Technology), anti-Slug (1:1,000; Cell Signaling Technology), and anti-β-actin (1:5,000; Sigma).

Xie H, & Lu X. (2023). circNFATC3 facilitated the progression of oral squamous cell carcinoma via the miR-520h/LDHA axis. Open Medicine, 18(1), 20230630.

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Western Blot Analysis of Metabolic Enzymes

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Cells were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors and centrifuged at 15,000× g for 15 min at 4 °C. Western blots were performed with 20–30 μg of cell extract. Proteins were separated in 8–12% SDS-PAGE and transferred to an Immobilon membrane (Merk Millipore, Burlington, MA, USA). Following primary antibodies were used: anti-phospho-Ser62/T58 c-Myc (Santa Cruz, Dallas, TX, USA; sc-377552), anti-c-Myc C-19 (Santa Cruz, Dallas, TX, USA; sc-788), anti-GLS1 (Abcam, Cambridge, UK; ab131554), anti-cSHMT A-2 (Santa Cruz, Dallas, TX, USA; sc-365203), anti-mSHMT F-11 (Santa Cruz, Dallas, TX, USA; sc-390641), anti-HK-2 (Cell Signaling, Danvers, MA, USA; 2867), anti-Glut1 (Santa Cruz, Dallas, TX, USA; sc-277228), anti-LDHA (Santa Cruz, Dallas, TX, USA; sc-137243). All membranes were normalized using mouse monoclonal anti-γ-tubulin antibody (Sigma-Aldrich, Darmstadt, Germany; T-6557). Horseradish peroxidase activity linked to secondary antibody was detected with ECL substrate (Pierce) in a Fujifilm LAS 3000 Intelligent Dark Box IV imaging system (Tokio, Japan).

Moreno-Felici J., Hyroššová P., Aragó M., Rodríguez-Arévalo S., García-Rovés P.M., Escolano C, & Perales J.C. (2019). Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2+. Cells, 9(1), 18.

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Immunohistochemical Analysis of Breast Cancer

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Antigen retrieval and IHC analyses were performed as described previously [32 (link)]. We used the following antibodies: anti-14-3-3ζ (Santa Cruz); anti-LDHA, anti-CREB, anti-phospho-CREB, anti-ERK, anti-phospho-ERK, anti-cleaved caspase 3 (Cell Signaling); and anti-MCM (Epitomics). For TMA, we used a 70-case, 208-core breast cancer tissue microarray (catalog no. BR208, US Biomax Inc.).

Chang C.C., Zhang C., Zhang Q., Sahin O., Wang H., Xu J., Xiao Y., Zhang J., Rehman S.K., Li P., Hung M.C., Behbod F, & Yu D. (2016). Upregulation of lactate dehydrogenase a by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression. Oncotarget, 7(23), 35270-35283.

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Protein Expression Analysis by Western Blot

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Lysates from cells were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride (PVDF) membranes (PerkinElmer, Boston, MA, USA), and blotted with primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibody. The primary antibodies used were anti-c-Myc and anti-HK2 (Abcam, Cambridge, MA, USA) and anti-PKM2 (Affinity Biosciences, OH, USA); and anti-LDHA, anti-GAPDH, and anti-actin (Santa Cruz, CA, USA); and anti-FBXW7 (ABclonal, Wuhan, China).

Zuo S., Wu L., Wang Y, & Yuan X. (2020). Long Non-coding RNA MEG3 Activated by Vitamin D Suppresses Glycolysis in Colorectal Cancer via Promoting c-Myc Degradation. Frontiers in Oncology, 10, 274.

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Quantifying Cell-Specific Protein Expression

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Samples obtained from all protocols were analyzed by Western blots with 4-12% SDS-PAGE (Invitrogen, NP0322BOX). Western blot assays were performed as described previously (Sherwood et al., 2010 (link)). The following primary antibodies were used for immunoblotting: anti-LDH-A (1/1000, Santa Cruz, sc- 27230), anti-LDH-B (1/500, Novus Biologicals, NB100-79987), and anti-LDH (1/1000, Santa Cruz, sc-133123). Antibodies were characterized in detail before being used for research (see the Supplemental Material). The blots were also probed with antibodies against cell-specific marker proteins: anti-GFAP (glial marker, 1/1000, Santa Cruz, sc-9065) and anti-SYP (presynaptic neuronal marker, 1/1000, Abcam, ab14692). For detection of antibodies, appropriate peroxidase-conjugated secondary antibodies were used in conjunction with enhanced chemiluminescence (Amersham Pharmacia Biosciences) to obtain images saved on film. The signals were quantitatively evaluated with Scion Image software. Equal protein loading was confirmed with anti-β-actin antibody (1/1000, Santa Cruz Biotechnology, sc-1616). Protein bands were scanned using an EPSON Perfection 4870 Photo scanner and quantitatively analyzed by Scion Image software.

Duka T., Anderson S.M., Collins Z., Raghanti M.A., Ely J.J., Hof P.R., Wildman D.E., Goodman M., Grossman L.I, & Sherwood C.C. (2014). SYNAPTOSOMAL LACTATE DEHYDROGENASE ISOENZYME COMPOSITION IS SHIFTED TOWARD AEROBIC FORMS IN PRIMATE BRAIN EVOLUTION. Brain, behavior and evolution, 83, 216-230.

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Immunohistochemical Analysis of Metabolic Markers

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Immunohistochemistry (IHC) was performed on human specimens and xenograft mouse tumors as described previously (Li et al., 2015b) using anti‐RAC1 (1 : 200 dilution; Abcam, Cambridge, UK), anti‐Ki67 (ZM0166, ready‐to‐use; ZSGB‐BIO, Beijing, China), anti‐PKM (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐LDHA (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti‐HK1 (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies according to the manufacturers’ instructions.

Zeng R., Zheng C., Gu J., Zhang H., Xie L., Xu L, & Li E. (2019). RAC1 inhibition reverses cisplatin resistance in esophageal squamous cell carcinoma and induces downregulation of glycolytic enzymes. Molecular Oncology, 13(9), 2010-2030.

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Protein Extraction and Western Blot Protocol

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Protein extraction and western blot were performed as described previously (Li et al., 2015b). Anti‐RAC1 (1 : 250 dilution) antibody was acquired from Cytoskeleton, Inc. (Denver, CO, USA). Anti‐PKM (1 : 500 dilution), anti‐LDHA (1 : 500 dilution), anti‐ALDOA (1 : 500 dilution), and anti‐HK1 (1 : 500 dilution) antibodies were obtained from Santa Cruz Biotechnology. Anti‐phospho‐AKT (Ser473) (1 : 1000 dilution), anti‐AKT (1 : 1000 dilution), anti‐phospho‐FoxO1 (Thr24)/FoxO3a (Thr32) (1 : 1000 dilution), anti‐FOXO3A (75D8) (1 : 1000 dilution), anti‐phospho‐S6 (Ser240/244) (1 : 1000 dilution), and anti‐S6 (5G10) (1 : 1000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

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Western Blot Analysis of Metabolic Regulators

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Cells were lysed by RIPA buffer, and 20 μg of total protein was separated and transferred onto NC membranes. The membranes were probed with the following primary antibodies: anti‐SETD1A (Bethyl, Montgomery, TX, USA, Cat#A300‐289A), anti‐HIF1α (BD, Franklin lake, NJ, USA, Cat#610959), anti‐HK2 (Santa Cruz, Santa Cruz, CA, USA, Cat#sc‐130358), anti‐LDHA (Santa Cruz, Cat#sc‐133123), anti‐PCNA (CST, Danvers, MA, USA, Cat#2586s), Flag (Sigma, Burlington, MA, USA, Cat#F1804), HA (Sigma, Cat#H9658), and anti‐β‐actin (Sigma, Cat#A5316) at 4 °C overnight. Membranes were then probed with appropriated horseradish peroxidase (HRP)‐conjugated secondary antibody and visualized by chemiluminescence.

Wu J., Chai H., Xu X., Yu J, & Gu Y. (2020). Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer. Molecular Oncology, 14(6), 1397-1409.

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Antibody Procurement for Cell Analysis

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Human anti-cleaved PARP and anti-flag antibodies were obtained from Cell Signaling Technology. Anti-tubulin was procured from Bioworld Company. Anti-GLUT1 and anti-LDHA antibodies were purchased from Santa Cruz Biotechnology. Anti-SUN2 (HPA001209) and anti-Sirt5 (HPA021798) antibodies were obtained from Sigma-Aldrich.

Lv X.B., Liu L., Cheng C., Yu B., Xiong L., Hu K., Tang J., Zeng L, & Sang Y. (2015). SUN2 exerts tumor suppressor functions by suppressing the Warburg effect in lung cancer. Scientific Reports, 5, 17940.

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