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Anti β actin

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Anti-β-actin is a primary antibody that specifically binds to the β-actin protein, which is a highly conserved and ubiquitously expressed cytoskeletal protein. It is commonly used as a loading control or reference marker in various biochemical and cellular analyses.

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Lab products found in correlation

Streptavidin hrp, by Cell Signaling Technology (2 mentions) Sab4200181, by Merck Group (2 mentions) Anti egr1, by Cell Signaling Technology (2 mentions) Sc 2324, by Santa Cruz Biotechnology (2 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions) Anti gfp, by GenScript (2 mentions) Supersep ace 15 precast gel, by Fujifilm (2 mentions) Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions) Clodronate liposome, by Yeasen (1 mentions) Anti mouse p mlkl, by Abcam (1 mentions) Influenza a virus np antibody, by GeneTex (1 mentions) Mouse tnf α elisa kit, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Takara Bio (1 mentions) Hiscript 2 q rt supermix for qpcr, by Vazyme (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Chamq universal sybr qpcr master mix, by Vazyme (1 mentions) Lats2 a300 479a, by Fortis Life Sciences (1 mentions) Ab27671, by Abcam (1 mentions) Anti ha antibody, by Cell Signaling Technology (1 mentions) Anti pmp70, by Merck Group (1 mentions)

13 protocols using anti β actin

1

Quantitative PCR and Cytotoxicity Assays

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HiScript II QRT SuperMix for qPCR (Vazyme, Nanjing, China), ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing), the lactate dehydrogenase (LDH) cytotoxicity assay kit (FUJIFILM, China). The following primary antibodies were used: Anti-mouse P-MLKL (1:1000, Abcam, Cambridge, MA, USA, ab196436), Anti-β-actin (1:1000, GenScript Biotechnology, Nanjing, China, A00702), Anti-human P-MLKL (1:1000, Cell Signaling Technology, Danvers, MA, USA, 91689 S), Influenza A Virus NP antibody (1:5000, GeneTex, Irvine, CA, USA, GTX125989). HRP anti-rabbit secondary antibody (1:8000, HuaAn Biotechnology, China, HA1001), HRP anti-mouse secondary antibody (1:8000, HuaAn Biotechnology, HA1006), Halt protease and phosphatase inhibitor cocktail (Selleck, USA), Trizol reagent (Takara, Kyoto, Japan), RIPA Lysis Buffer (Beyotime Biotechnology, China). Mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA, USA), Mouse MCP-1 ELISA kit (Invitrogen), Mouse TNF-α ELISA kit (eBioscience, San Diego, CA, USA), Mouse OPN ELISA kit (Multi Sciences, China), Human OPN ELISA kit (Multi Sciences), Clodronate liposomes (YEASEN, China).
Wang J., Li X., Wang Y., Li Y., Shi F, & Diao H. (2022). Osteopontin aggravates acute lung injury in influenza virus infection by promoting macrophages necroptosis. Cell Death Discovery, 8, 97.
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2

Antibodies for YAP, Lats, and CTGF

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Antibodies for YAP (#4912) and Phospho-YAP (S127; #4911) were purchased from Cell Signaling Technology. The Lats1 (A300–477A) and Lats2 (A300–479A) antibodies used in immunoprecipitation were from Bethyl Laboratories. TAZ (HPA007415) antibody was obtained from Sigma-Aldrich. Anti-TEF (610922) was purchased from BD Transduction Laboratories. Antibodies for ETAR (SC33535), Gαq/11 (SC392), and CTGF (SC14939) were from Santa Cruz Biotechnology. Anti-β-actin (A00702) was purchased from GenScript. Antibody for GS was from Abcam. All antibodies were used properly following manufacturer-recommended dilutions in immunoblotting and immunofluorescence staining analysis.
Wang Z., Liu P., Zhou X., Wang T., Feng X., Sun Y.P., Xiong Y., Yuan H.X, & Guan K.L. (2017). Endothelin Promotes Colorectal Tumorigenesis by Activating YAP/TAZ. Cancer research, 77(9), 2413-2423.
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3

Immunoblotting Antibody Detection Protocol

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Detecting reagents, including streptavidin-HRP (1:2,000 for WB, 3999S), anti-Myc antibody (1:2,000 for WB, 2276S), anti-Myc HRP (1:1,000 for WB, 2040S), anti-HA antibody (1:2,000 for WB, 2367S), anti-HA HRP (1:1,000 for WB, 2999S), V5 (1:1,000 for immunofluorescence, 13202S), and anti-GFP HRP (1:1,000 for WB, 2037S) were purchased from Cell Signaling Technology. Antibodies against V5 (1:3,000 for WB, ab27671) and MARCH5 (1:2,000 for WB, ab174959) were purchased from Abcam. Anti-GDAPH (1:4,000 for WB, A00191) and anti–β-actin (1:4,000 for WB, A00702) were purchased from GenScript. Other antibodies used in this study included anti-FLAG (1:2,000 for WB, GNI14110-FG) and anti-FLAG HRP (GNI4310-FG; GNI), anti-PMP70 (1:3,000 for WB, SAB4200181; Sigma-Aldrich), and anti-GFP (1:3,000 for WB, M20004; Abmart). Anti–V5-HRP (1:5,000 for WB, R961-25) was purchased from Thermo Fisher Scientific. Other primary antibodies used were anti-MARCH5 (19168S; Cell Signaling Technology), anti-PEX19 (14713–1-AP; Proteintech), anti-PEX3 (sc-271477; Santa Cruz Biotechnology), anti-catalase (219010; Millipore), and anti-PEX13 (ab235043; Abcam). Secondary antibodies conjugated to HRP were purchased from GenScript.
Zheng J., Chen X., Liu Q., Zhong G, & Zhuang M. (2021). Ubiquitin ligase MARCH5 localizes to peroxisomes to regulate pexophagy. The Journal of Cell Biology, 221(1), e202103156.
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4

Western Blot Analysis of EGR1 Expression

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Protein isolates (20 μg) were resolved on 7.5% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels before transfer to polyvinylidene difluoride (PVDF) membranes. After protein transfer, PVDF membranes were blocked for 1 h with 5% non-fat dry milk ((sc-2324 (Blotto)) Santa Cruz Biotechnology Inc.) in Tris-buffered saline with Tween 20 (TBS-T) before incubation overnight at 4°C with the following primary antibodies: anti-EGR1 (#4154, Cell Signaling Technology, Inc.) diluted 1:1000 and anti-β-actin (#A00702, GenScript Biotech, Piscataway, NJ) diluted 1:100,000 in 5% non-fat milk in TBS-T buffer. Following primary antibody incubation, immunoblots were probed with anti-rabbit ((A27036 (1:5000 dilution)) ThermoFisher Scientific Inc.) and anti-mouse IgG secondary antibodies conjugated with horse radish peroxidase (HRP ((#7076 (1:10,000 dilution)) Cell Signaling Technology, Inc.) respectively in 5% non-fat milk in TBS-T buffer for 1 h at room temperature. The resultant chemiluminescence was detected with the SuperSignal West Pico PLUS Chemiluminescent Substrate ((#1863097) ThermoFisher Scientific, Inc.). Immunoreactive bands were digitally imaged using the Azure 300 Chemiluminescent Western Blot Imager (Azure Biosystems, Inc., Dublin, CA).
Maurya V.K., Ying Y., Lanza D.G., Heaney J.D, & Lydon J.P. (2023). A CRISPR/Cas9-engineered mouse carrying a conditional knockout allele for the early growth response-1 transcription factor. Genesis (New York, N.Y. : 2000), 61(3-4).
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5

Western Blot Analysis of GFP

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The cells were plated and washed with phosphate-buffered saline before collection.
Proteins from the cultured cells were processed using SuperSep Ace 15% precast gel (FUJIFILM Wako Pure Chemical Co.) and transferred on to a polyvinylidene fluoride membrane. The membranes were probed with anti-GFP (0.5 µg/mL, chicken polyclonal; Genscript, Piscataway, NJ, USA) and anti-β-actin (1 µg/mL, mouse monoclonal, clone. AC-15; Sigma-Aldrich) antibodies. Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were then added. Horseradish peroxidase (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Mokuda S., Watanabe H., Kohno H., Ishitoku M., Araki K., Hirata S., & Sugiyama E. (2022). N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes.
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6

Protein Detection Reagents for Western Blotting and Immunofluorescence

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Detecting reagents used for western blotting include: streptavidin-HRP (Cell Signaling, 3999S, 1:3000), anti-Myc (Cell Signaling, 2276S, 1:3000), anti-V5 (Abcam, ab27671, 1:3000), anti-β actin (GenScript, A00702, 1:3,000), anti-FLAG (GNI, GNI14110-FG, 1:3000), anti-ABCD3 (Sigma, P0497, 1:3000), anti-MFN1 (Cell Signaling, D6E2S, 1:3000), anti-MFN2 (Cell Signaling, D1E9, 1:3000). Antibodies used for immunofluorescence include: anti-MFN1 (Cell Signaling, D6E2S, 1:500), anti-MFN2 (Cell Signaling, D1E9, 1:500), anti-calnexin (Abcam, ab22595, 1:500), anti-EEA1 (Cell Signaling, C45B10, 1:500), anti-GM130 (Abcam, ab52649, 1:500), anti-LAMP1 (Cell Signaling, D2D11, 1:500), anti-ABCD3 (Sigma, SAB4200181, 1:500), anti-ABCD3 (Sigma, P0497, 1:500), anti-catalase (Merck/Millipore, 219010, 1:500), anti-FLAG (GNI, GNI14110-FG, 1:500), anti-FLAG (Proteintech, 20543-1-AP, 1:500), anti-Myc (Cell Signaling, 2276S, 1:500), anti-COX4 (Proteintech, 11242-1-AP, 1:500).
Huo Y., Sun W., Shi T., Gao S, & Zhuang M. (2022). The MFN1 and MFN2 mitofusins promote clustering between mitochondria and peroxisomes. Communications Biology, 5, 423.
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7

Histone-Specific Western Blotting Protocol

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For histone-specific Western blotting, histones were enriched by lysing cells in an acid lysis buffer (10 nM HEPES, 1.5 mM MgCl2, 10 mM KCl). For all other Western blots protein lysates were generated using RIPA Buffer (Cell Signaling, #9806). Protein lysates and histone extractions were separated by SDS-PAGE, transferred to a PVDF membrane, and detected by Western blotting (WB). Membranes were blocked with 5% BSA in 1X TBS with 0.01% Tween20 (TBST) for 1 h at room temperature and incubated with primary antibodies in 5% BSA in TBST overnight at 4 C (1:1,000 anti-EZH2 (Cell Signaling, D2C9), 1:1,000 anti-EZH1 (Proteintech, #20852-1AP); 1:1,000 anti-EZH1 (Reinberg lab21 (link)), 1:1,000 anti-H3K27me3 (EMD Millipore, 07-449), 1:5,000; anti-β-actin (GenScript, A00702), 1:10,000 anti-H4 (Abcam, ab10158)). The next day the membrane was washed three times with TBST, incubated for 1 h with the corresponding secondary anti-Rabbit HRP (Invitrogen, #31458) and anti-Mouse HRP (Invitrogen, #SA1-100), and washed three times with TBST. The membranes were developed using Pierce™ ECL Western Blotting Substrate kit (Thermo Scientific, #32106) and exposed to autoradiography films following development in AFP Mini-Med 90 X-Ray Fil Processor. ImageJ software was used for densitometry quantification of WB bands.
Gracia-Diaz C., Zhou Y., Yang Q., Maroofian R., Espana-Bonilla P., Lee C.H., Zhang S., Padilla N., Fueyo R., Waxman E.A., Lei S., Otrimski G., Li D., Sheppard S.E., Mark P., Harr M.H., Hakonarson H., Rodan L., Jackson A., Vasudevan P., Powel C., Mohammed S., Maddirevula S., Alzaidan H., Faqeih E.A., Efthymiou S., Turchetti V., Rahman F., Maqbool S., Salpietro V., Ibrahim S.H., di Rosa G., Houlden H., Alharbi M.N., Al-Sannaa N.A., Bauer P., Zifarelli G., Estaras C., Hurst A.C., Thompson M.L., Chassevent A., Smith-Hicks C.L., de la Cruz X., Holtz A.M., Elloumi H.Z., Hajianpour M.J., Rieubland C., Braun D., Banka S., French D.L., Heller E.A., Saade M., Song H., Ming G.L., Alkuraya F.S., Agrawal P.B., Reinberg D., Bhoj E.J., Martínez-Balbás M.A, & Akizu N. (2023). Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders. Nature Communications, 14, 4109.
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8

Investigating ER-associated proteins in ORMDL3

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The following antibodies were used: anti-SPTLC1 (Sigma-Aldrich, HPA063907), anti-SPTLC2 (Abcam, ab236900), polyclonal anti-ORMDL3 (MilliporeSigma, ABN417) that detects ORMDL1, -2, and -3 (22 (link)), mouse anti-FLAG (immunofluorescence; Sigma-Aldrich, F1804), rabbit anti-FLAG (immunoblot; Proteintech, 20543-1-AP), anti-VAPB (Proteintech, 14477-1-AP), anti-UBB (Cell Signaling Technology, 3933), anti–β-actin (GenScript, A00702), anti-TOMM40 (Proteintech, 18409-1-AP), anti-HSPA5/BiP (Abcam, ab21685); and the secondary antibodies anti-mouse–Alexa Fluor 488 (Invitrogen, A-11029), anti-mouse–HRP (Jackson ImmunoResearch, 115-035-146), and anti-rabbit–HRP (Jackson ImmunoResearch, 111-035-003).
Lone M.A., Aaltonen M.J., Zidell A., Pedro H.F., Morales Saute J.A., Mathew S., Mohassel P., Bönnemann C.G., Shoubridge E.A, & Hornemann T. (2022). SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins. The Journal of Clinical Investigation, 132(18), e161908.
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9

Western Blot Analysis of EGR1 and β-Actin

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Protein (20 μg) from cell lysates was resolved on 4-15% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels before transfer to polyvinylidene difluoride (PVDF) membranes. Following protein transfer, PVDF membranes were blocked for 1 hour with 5% non-fat dry milk ((sc-2324 (Blotto)) Santa Cruz Biotechnology Inc., Dallas, Texas) in Tris-buffered saline with Tween 20 (TBS-T) and incubated overnight at 4°C with the following primary antibodies: anti-EGR1 (#4154, Cell Signaling Technology, Inc., Danvers, MA) diluted 1:1000 and anti-β-actin (#A00702, GenScript Biotech, Piscataway, NJ) diluted 1:100000 in 5% non-fat milk in TBS-T. Blots were then probed with anti-rabbit ((A27036 (1:5000 dilution)) ThermoFisher Scientific Inc.) and anti-mouse IgG secondary antibodies conjugated with HRP ((#7076 (1:10000 dilution)) Cell Signaling Technology, Inc.) respectively in 5% non-fat milk in TBS-T for 1 hour at room temperature. Chemiluminescence was detected with the SuperSignal West Pico PLUS Chemiluminescent Substrate ((#1863097) ThermoFisher Scientific, Inc.). Immunoreactive bands were digitally imaged using the Bio-Rad ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA).
Maurya V.K., Szwarc M.M., Fernandez-Valdivia R., Lonard D.M., Song Y., Joshi N., Fazleabas A.T, & Lydon J.P. (2022). Early growth response 1 transcription factor is essential for the pathogenic properties of human endometriotic epithelial cells. Reproduction (Cambridge, England), 164(2), 41-54.
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10

Western Blot Analysis of Protein Markers

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Lytic samples were run on 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 8% native-PAGE, transferred to polyvinylidene fluoride membranes and probed with anti-GOLPH2 (1:1000, Abcam, ab109628), anti-P53 (1:1000, CST, 2524S), anti-p-P53 (Ser 315) (1:1000, Bioss, bs-3704R), anti-GAPDH (1:1000, Abkine, A01020), and anti-β-actin (1:500, Genscript, A00730). After incubating with anti-rabbit or anti-mouse IgG secondary antibodies membranes were conjugated to horseradish peroxidase at a dilution of 1:5000 and visualized with an enhanced chemiluminescence system (Bio-Rad, Hercules, CA, USA).
Song Q., He X., Xiong Y., Wang J., Zhang L., Leung E.L, & Li G. (2021). The functional landscape of Golgi membrane protein 1 (GOLM1) phosphoproteome reveal GOLM1 regulating P53 that promotes malignancy. Cell Death Discovery, 7, 42.
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