The following primary antibodies were used for this study: rabbit anti‐VAPA (homemade #1006‐04; Teuling et al, 2007 ) and anti‐VAPB (homemade #1006‐00; Teuling et al, 2007 ); mouse anti‐synaptotagmin (SySy, 105311, clone 604.2); rabbit anti‐SCRN1 (SySy, 289003; used in Fig 1 E), rabbit anti‐SCRN1 (Abcam, ab105355; used in Fig EV1 D), and rabbit anti‐SCRN1 (Sigma, HPA024517, RRID:AB_2184811; used for all other experiments; validation SCRN1 antibodies in Fig EV1 G–J); guinea pig anti‐vGlut (Millipore, ab5095); rat anti‐HA (Roche, 1867423; used for immunostainings); mouse anti‐HA (BioLegend, mms‐101p, clone 16B12; used for immunoblots); mouse anti‐actin (Chemicon, MAB1501R, clone C4); rabbit anti‐GFP (Abcam, ab290); mouse anti‐Myc (Santa Cruz, SC40, clone 9E10); and mouse α‐Tubulin (Sigma, T5168, clone B‐5‐1‐2, RRID:AB_477579). The following secondary antibodies were used for this study: anti‐rabbit Alexa 488 (Life Technologies, A11034), anti‐rabbit Alexa 568 (Life Technologies, A11036), anti‐rat Alexa 568 (Life Technologies, A11077), anti‐guinea pig Alexa 568 (Life Technologies, A11075), anti‐mouse Alexa 647 (Life Technologies, A21236), anti‐mouse anti‐HRP (Dako, P0260), anti‐rabbit anti‐HRP (Dako, P0399), anti‐mouse IRDye 680LT (Li‐Cor, 926‐68020), and anti‐rabbit IRDye 800CW (Li‐Cor, 926‐32211).
Sc 40
Manufactured by Merck Group
The Sc-40 is a laboratory equipment product manufactured by Merck Group. It is a specialized instrument designed for scientific research and analysis. The core function of the Sc-40 is to perform precise measurements and data collection, supporting various laboratory applications. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit alexa 568, by Thermo Fisher Scientific (1 mentions)
T5168, by Merck Group (1 mentions)
Anti rabbit irdye 800cw, by LI COR (1 mentions)
Ab290, by Santa Cruz Biotechnology (1 mentions)
Mms 101p, by BioLegend (1 mentions)
Fluorchem hd2 imager, by Bio-Techne (1 mentions)
Supersignal west pico chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Southern Biotech (1 mentions)
β tubulin, by Merck Group (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
F7425, by Abcam (1 mentions)
Ab8224, by Abcam (1 mentions)
Ab52866, by Abcam (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
F1804, by Merck Group (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Na934v, by GE Healthcare (1 mentions)
6 protocols using sc 40
1
Antibody Panel for Synaptic Protein Analysis
2
Western Blot and Immunofluorescence Analysis of Signaling Proteins
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Membranes were probed with primary antibodies for phosphorylated ERK (Cell Signaling Technology; 4370), total ERK (Santa Cruz Biotechnology; sc-153 or Cell Signaling Technology; 4696), sheep polyclonal anti-DUSP5 (13 (link)), rabbit anti-DUSP5 (Abcam; ab200708), c-Myc (Santa Cruz Biotechnology; sc-40), β-tubulin (Sigma; T8328), or α-tubulin (Santa Cruz Biotechnology; sc-23948 HRP). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech) were used at a concentration of 1:2,000. SuperSignal West Pico chemiluminescence system (Thermo Scientific) and a FluorChem HD2 Imager (Alpha Innotech) were used to detect proteins. For indirect immunofluorescence, anti-DUSP5 antibody (Abcam) was diluted 1:100 in phosphate-buffered saline with 0.1% Tween-20 (PBS-T) containing 1% BSA and incubated overnight. Secondary goat anti-rabbit IgG conjugated to Alexa Fluor 594 (Thermo Fisher; A-11012) and DAPI were used for detection of DUSP5 and nuclei, respectively.
3
Western blot analysis of PARN
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Cells were dispersed in ice-cold Hypotonic Lysis Buffer, lysed by sonication, and spun at 15,000 rpm at 4°C for 10 min to pellet insoluble debris. Thirty to fifty micrograms of the cleared lysate was separated on a 10% SDS-polyacrylamide gel and electrotransferred to an Immobilon-P PVDF membrane (EMD Millipore). The primary antibodies used in this study include rabbit anti-PARN (Abcam, ab27778), mouse anti-myc (9E10; Santa Cruz Biotechnology, sc-40), rabbit anti-Flag (Sigma, F7425), rabbit anti-α-tubulin (Abcam, ab52866), and mouse anti-β-actin (Abcam, ab8224).
4
Western Blot Analysis of Spliceosomal Complexes
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Twenty micrograms of lysates or total eluates obtained from CoIP or GST pulldown experiments were separated by SDS–PAGE and transferred electrophoretically to Immobilon-P membranes (Millipore). Membranes were blocked in 5% nonfat dry milk in PBS-Tween (0.1% Tween20 (Sigma-Aldrich)) and incubated with anti-EFTUD2 (1:10,000, Abcam; ab72456), anti-PRPF8 (1:5,000, Abcam; ab87433), anti-SNRNP200 (1:2,000, Abcam; ab118713), anti-CD2BP2 (1:2,000, Abcam; ab136141), anti-PRPF6 (1:5,000, Abcam; ab99292), anti-DDX23 (1:2,000, Abcam; ab70461), anti-SNRNP40 (1:2,000, Abcam; ab155592), anti-AAR2 (1:2,000, Abcam; ab150727), anti-RUVBL2 (1:2,000, Abcam; ab36569), anti-TSC1 (1:2,000; Thermo Scientific; 37-0400), anti-TSC2 (1:2,000, Santa Cruz; SC-893), anti-GST (1:5,000, Abcam; ab9085), anti-6 × His (1:5,000, Abcam; ab18184), anti-Myc (1:5,000, Santa Cruz; SC-40) and anti-FLAG M2 (1:5,000, Sigma-Aldrich; F1804). Protein bands were visualized using anti-rabbit and anti-mouse IgG secondary antibodies linked to horseradish peroxidase (1:2,500, GE Healthcare; NA934V and NA931V) and ECL prime (GE Healthcare). See Supplementary Figs 3 and 4 for uncropped versions of each blot.
5
Myc-Tagged Protein Immunoprecipitation
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HEK293T cells (ATCC, CRL1573) were transfected with expression vectors using Lipofectamine 2000 (Invitrogen), and cultured for 24 h. The cells were washed with phosphate-buffered saline at room temperature, and lysed in ice-cold lysis buffer consisting of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1× protease inhibitor cocktail (Sigma-Aldrich). Immunoprecipitation was performed using 10 µl, monoclonal anti-myc agarose beads (Pierce, #20168) according to the manufacturer’s recommendation. After 2 h of incubation at 4 °C, immunoprecipitated proteins were washed five times with washing buffer (a modified lysis buffer containing 500 mM NaCl), and separated by polyacrylamide gel electrophoresis, then transferred to nitrocellulose membrane, probed with 9E10 anti-myc (1:1000, Santa Cruz Biotechnology, sc-40) or anti-Flag (1:3000, Sigma-Aldrich, F3165) antibody, and detected using the Western-Lightning Plus-ECL substrate (PerkinElmer). All uncropped western blots can be found in Supplementary Fig. 6 .
6
Regulation of Runx1 by c-Myb
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293T cells were grown in DMEM supplemented with 10% bovine calf serum. Cell transfection, cell extracts preparation, immunoprecipitation, and western Blot have been described (42 (link)). Constructs with Myc-tagged zebrafish c-myb and Flag-tagged zebrafish runx1 were transfected into 293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitants were examined by western blot using anti-MYC and anti-FLAG antibodies. Input represents 10% of total cell lysates used for immunoprecipitation. Anti-MYC and anti-FLAG antibodies were obtained from Santa Cruz (sc-40) and Sigma (ab6658), respectively.
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