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Biotin is a water-soluble vitamin that plays a crucial role in various metabolic processes within cells. It functions as an essential cofactor for several carboxylase enzymes, which are involved in the synthesis of fatty acids, the metabolism of amino acids, and the production of energy. Biotin is a widely used tool in biochemistry and molecular biology for its ability to form a strong, non-covalent interaction with the protein avidin or streptavidin, enabling numerous applications in affinity-based techniques and assays.

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Lab products found in correlation

Flag tag (flag), by Merck Group (3 mentions) Nedd8, by Cell Signaling Technology (2 mentions) Hnrnp e1, by Abnova (2 mentions) K63 ubiquitin, by Cell Signaling Technology (2 mentions) Hsp90, by Santa Cruz Biotechnology (2 mentions) K48 ubiquitin, by Cell Signaling Technology (2 mentions) Flag tag (flag), by Cell Signaling Technology (2 mentions) E cadherin, by Cell Signaling Technology (2 mentions) Vimentin, by Cell Signaling Technology (2 mentions) Chemidoc system, by Bio-Rad (2 mentions) N cadherin, by BD (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Rela p65, by Cell Signaling Technology (1 mentions) Anti rabbit igg, by Cell Signaling Technology (1 mentions) Anti mouse igg, by Cell Signaling Technology (1 mentions) Caspase 8, by Cell Signaling Technology (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Phospho tbk1, by Cell Signaling Technology (1 mentions) Nf κb1 p105 p50, by Cell Signaling Technology (1 mentions) Phospho p65 rela, by Cell Signaling Technology (1 mentions)

9 protocols using biotin

1

Horseradish Peroxidase-conjugated Antibodies for Western Blotting

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The following horseradish peroxidase (HRP)-conjugated antibodies were used in this study: FLAG (Sigma–Aldrich, A8592, 1:5,000), AGIA19 (link) (produced in our laboratory, 1:10,000), α-tubulin (MBL, PM054-7, 1:10,000), and biotin (Cell Signaling Technology, #7075, 1:5,000). The following primary antibodies were used in this study: CRBN (#71810, 1:1000), IκBα (#4814, 1:1000), phospho-IκBα (#9246, 1:1000), NF-κB1 p105/p50 (#12540, 1:1000), p65 (RelA) (#8242, 1:1000), phospho-p65 (RelA) (#3033, 1:1000), CYLD (#8462, 1:1000), RIP1 (#3493, 1:1000), caspase-8 (#9746, 1:1000), caspase-3 (#9662, 1:1000), PARP (#9542, 1:1000), FADD (#2782, 1:1000), phospho-TBK1 (#5483, 1:1000), and TBK1 (#3504, 1:1000) (all from Cell Signaling Technology); and lamin B (#sc-6217, 1:500) (from Santa Cruz Biotechnology). Anti-rabbit IgG (HRP-conjugated, Cell Signaling Technology, #7074, 1:10,000), anti-mouse IgG (HRP-conjugated, Cell Signaling Technology, #7076, 1:10,000), and anti-goat IgG (HRP-conjugated, Invitrogen/Thermo Fisher Scientific, #81-1620, 1:10,000) were used as secondary antibodies.
Yamanaka S., Shoya Y., Matsuoka S., Nishida-Fukuda H., Shibata N, & Sawasaki T. (2020). An IMiD-induced SALL4 degron system for selective degradation of target proteins. Communications Biology, 3, 515.
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2

Immunoblot Analysis of Protein Interactions

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Immunoblot analysis was performed as described previously 47 (link). Briefly, cells were lysed in RIPA buffer and denatured at 95°C for 5 min in Laemmli buffer. Samples were resolved on SDS–PAGE gels and transferred to PVDF membrane. Blots were probed with antibodies specific to ARIH1 (VPA00397; BioRad), hnRNP E1 (M01; Abnova), E-cadherin (3195; Cell Signaling), N-cadherin (BD Transductions), Vimentin (5741; Cell Signaling), Biotin (5597; Cell Signaling), Flag (14793; Cell Signaling), K48 Ubiquitin (8081; Cell Signaling), K63 Ubiquitin (5621; Cell Signaling), Nedd8 (2745; Cell Signaling), Hsp90 (sc13119: Santa Cruz Biotechnology), CD44 (GTX102111; GeneTex), V5 (R96025; ThermoFisher), UPF1 (VMA00627; BioRad), Filamin-A (VMA00322; BioRad), Cortactin (VMA00430; BioRad), CAND1 (VMA00610; BioRad), and FASN (VMA00266; BioRad). Chemiluminescence was detected by CCD camera (BioRad ChemiDoc system).
Howley B.V., Mohanty B., Dalton A., Grelet S., Karam J., Dincman T, & Howe P.H. (2022). The ubiquitin E3 ligase ARIH1 regulates hnRNP E1 protein stability, EMT and breast cancer progression. Oncogene, 41(12), 1679-1690.
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3

Immunoblot Analysis of Cellular Proteins

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Immunoblot analysis was performed as described previously [47 (link)]. Briefly, cells were lysed in RIPA buffer and denatured at 95 °C for 5 min in Laemmli buffer. Samples were resolved on SDS–PAGE gels and transferred to PVDF membrane. Blots were probed with antibodies specific to ARIH1 (VPA00397; BioRad), hnRNP E1 (M01; Abnova), E-cadherin (3195; Cell Signaling), N-cadherin (BD Transductions), Vimentin (5741; Cell Signaling), Biotin (5597; Cell Signaling), Flag (14793; Cell Signaling), K48 Ubiquitin (8081; Cell Signaling), K63 Ubiquitin (5621; Cell Signaling), Nedd8 (2745; Cell Signaling), Hsp90 (sc13119: Santa Cruz Biotechnology), CD44 (GTX102111; GeneTex), V5 (R96025; ThermoFisher), UPF1 (VMA00627; BioRad), Filamin A (VMA00322; BioRad), Cortactin (VMA00430; BioRad), CAND1 (VMA00610; BioRad), and FASN (VMA00266; BioRad). Chemiluminescence was detected by CCD camera (BioRad ChemiDoc system).
Howley B.V., Mohanty B., Dalton A., Grelet S., Karam J., Dincman T, & Howe P.H. (2022). The ubiquitin E3 ligase ARIH1 regulates hnRNP E1 protein stability, EMT and breast cancer progression. Oncogene, 41(12), 1679-1690.
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4

Immunoblot Detection of Tagged Proteins

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The following HRP-conjugated antibodies were used in this study: FLAG (Sigma-Aldrich, A8592, MBL, M185-7), AGIA (produced in our laboratory)41 (link), α-tubulin (MBL, PM054-7), and biotin (Cell Signaling Technology, #7075). To detect the proteins spotted on the array, anti-DDDDK-tag mAb-Alexa Fluor 647 (MBL, M185-A64) was used.
Sugiyama S., Yamada K., Denda M., Yamanaka S., Ozawa S., Morishita R, & Sawasaki T. (2022). CF-PPiD technology based on cell-free protein array and proximity biotinylation enzyme for in vitro direct interactome analysis. Scientific Reports, 12, 10592.
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5

Comprehensive Western Blot Analysis

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Cells were lysed in PBS solution supplemented with 1X radioimmunoprecipitation assay buffer (Millipore) and 2X Halt protease-phosphatase inhibitor cocktail (Thermo Fisher Scientific). Upon clearance, samples were analyzed by SDS-PAGE, electrotransferred to nitrocellulose membranes (Invitrogen), and blocked by 5% powdered milk in PBSt (PBS supplemented with 0.1% tween) solution. Development was conducted with ECL reagent (Thermo Fisher Scientific) and imaging was performed with the ChemiDoc ZRS+ imager (Bio-Rad). Antibodies used for Western blot analysis include IRE1α (3294, rabbit; Cell Signaling Technology), β-actin (5125, rabbit; Cell Signaling), GAPDH (97166, mouse; Cell Signaling), ATF6 (66563-1-Ig, mouse; ProteinTech), TAP1 (12341, rabbit; Cell Signaling), CHOP (2895, mouse; Cell Signaling), ATF4 (11815, rabbit; Cell Signaling), ovalbumin (P1-196, rabbit; Thermo Fisher Scientific), human Fc (ab977225, rabbit; Abcam), Myc tag (2272, rabbit; Cell Signaling), and biotin (5597, rabbit; Cell Signaling). IRE1α lumenal domain (mouse), XBP1s (rabbit), and pIRE1α (rabbit) antibodies were generated at Genentech. Secondary antibodies used were for mouse (715-035-150; The Jackson Laboratory) and rabbit (711-035-152; The Jackson Laboratory).
Guttman O., Le Thomas A., Marsters S., Lawrence D.A., Gutgesell L., Zuazo-Gaztelu I., Harnoss J.M., Haag S.M., Murthy A., Strasser G., Modrusan Z., Wu T., Mellman I, & Ashkenazi A. (2022). Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation. The Journal of Cell Biology, 221(6), e202111068.
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6

Protocol for Proteomics in PC-3 Cells

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PC-3 cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS. The following antibodies were used: BIOTIN (Cat. 5597) and GAPDH (Cat. 2118) were from Cell Signaling; CLU (Cat. 05-354) and ACTIN (Cat. MAB1501) were from Millipore; FOS (Cat. sc-166940), TIA-1 (sc-1751) and HSPA1A (cat. sc-32239) were from Santa Cruz Biotech; JUN (Cat. 610326) and G3BP1 (Cat. 611127BD) were from BD Biosciences; BAX (Cat. ab32503, ab77566), APX2 (Cat. ab222414) and YB-1 (Cat. ab76149) were from Abcam; HIF1A (Cat. NB100-131) was from Novus; Fluorescently labeled secondary antibodies (mouse, Alexa Fluor 488/594; rabbit, Alexa Fluor 488/594; and goat, Alexa Fluor 488/594), TRIzol, RNAiMAX transfection reagent, Dynabeads M-280 Streptavidin, DMEM, FBS, Click-iT Protein Reaction Buffer Kit, BIOTIN-alkyne and l-azidohomoalanine (AHA) were from Life Technologies; Trolox, BIOTIN-tyramide, sodium ascorbate, sodium deoxycholate and cycloheximide were from Sigma; DMEM without l-lysine and l-arginine was from Caisson Labs (USA); 13C6-arginine and D4-lysine were from Silantes; FluorSave was from Merck; and G3BP1-APEX and CTRL-APEX constructs were custom made from GenScript.
Somasekharan S.P., Zhang F., Saxena N., Huang J.N., Kuo I.C., Low C., Bell R., Adomat H., Stoynov N., Foster L., Gleave M, & Sorensen P.H. (2020). G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress. Nucleic Acids Research, 48(12), 6855-6873.
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7

Immunoblotting Assay for Cell Signaling

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Cells were collected and lysed in RIPA lysis buffer (Cat. # BP-115DG, Boston Bio Products, Ashland, MA, USA) supplemented with protease and phosphatase inhibitor cocktails (Cat. # PPC1010, Sigma-Aldrich, St. Louis, MO, USA). Protein samples were made and immunoblotting was performed as described previously5 (link),48 (link). Antibodies purchased from Cell Signaling Technologies (CST) and the dilutions are as follows: Biotin (Cat. No. 5571S, 1:1000), PARP (Cat. No. 9542S, 1:1000), c-Myc (Cat. No. 5605S, 1:1000), MCL1 (Cat. No. 5453S, 1:1000), and ALK (Cat. No. 3791S, 1:1000), TRAF6 (Cat. No. 8082S, 1:1000). KEAP1 (Cat. No. 10503–2-AP, 1:1000) and NRF2 (Cat. No. 16396–1-AP, 1:1000) antibodies were purchased from Proteintech (Cat. No. 10503–2-AP, 1:1000). CDK9 antibody was purchased from Santa Cruz (Cat. No. sc-13130, 1:500). V5-tag antibody was purchased from Bethyl (Cat. No. A190–120P, 1:1000). TRIP12 antibody was purchased from Fortis (Cat. No. A301–814A-M, 1:1000). β-actin antibody was purchased from MP Biomedicals (Cat. No. 8691001, 1:20 000).
Pei J., Xiao Y., Liu X., Hu W., Sobh A., Yuan Y., Zhou S., Hua N., Mackintosh S.G., Zhang X., Basso K.B., Kamat M., Yang Q., Licht J.D., Zheng G., Zhou D, & Lv D. (2023). Piperlongumine (PL) conjugates induce targeted protein degradation. Cell chemical biology, 30(2), 203-213.e17.
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8

Antibody Detection Protocol for Protein Analysis

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The following HRP-conjugated antibodies were used in this study: FLAG (Sigma-Aldrich, A8592,1:5000), AGIA (produced in our laboratory), α-tubulin (MBL, PM054-7, 1:5000), GST (M071-7, 1:5000), and biotin (Cell Signaling Technology, #7075, 1:1000). The following primary antibodies were used in this study: IκBα (CST L35A5, 1:1000), RelA (CST L8F6, 1:1000), GFP (MBL, M048-3 1:1000), HSP90 (CST, #4874, 1:1000), Ra mAb (produced in our laboratory), and GATS (produced in our laboratory, 0.2 µg/mL). Anti-rabbit IgG (HRP-conjugated, Cell Signaling Technology, #7074, 1:10,000) and anti-mouse IgG (HRP-conjugated, Cell Signaling Technology, #7076, 1:10,000) were used as secondary antibodies.
Yamada K., Soga F., Tokunaga S., Nagaoka H., Ozawa T., Kishi H., Takashima E, & Sawasaki T. (2023). GATS tag system is compatible with biotin labelling methods for protein analysis. Scientific Reports, 13, 10243.
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9

Antibody Identification and Validation

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Antibodies used in this study include Flag (Sigma F7425), HA (Biolegend, 901501), GFP (1:1000, Abcam, ab13970), MBP (custom made), RAD51 (14B4, Abcam), RADX (NBP2, Novus), BRCA2 (OP95, Calbiochem), γH2AX (JBW301, Millipore), Biotin (Cell Signaling #5597), IdU (Abcam Cat#ab6326), and CldU (BD Cat#347580).
Mohamed T., Adolph M.B, & Cortez D. (2022). Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability. The Journal of Biological Chemistry, 298(3), 101672.
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