HeLa transfectants transiently expressing AQP11mycFlag and either Halo-RDEL, HaloGpx8 TM [22 ], or HaloAQP11 were grown and lysed as above. Postnuclear fractions were quantified and 1 mg of total protein incubated o/n at 4 °C with commercial anti-Flag crosslinked beads (Sigma). Next, beads were washed three times with a buffer containing 0.25% NP40 150 mM NaCl 10 mM Tris pH 7.4 and boiled in standard protein sample buffer. Aliquots of the postnuclear lysates were run with those immunoprecipitated samples in reducing SDS-PAGE, followed for Western blot with rabbit anti-Halo (Promega) and mouse anti-Flag antibodies (Sigma). Images were acquired using a Typhoon FLA-9000 (GE HealthCare) and processed with ImageJ.
Rabbit anti halo
Manufactured by Promega
Rabbit anti-Halo is an antibody that binds to the HaloTag® protein, a versatile protein tag used for labeling and purification of recombinant proteins. The Rabbit anti-Halo antibody can be used to detect the presence and track the localization of HaloTag-fusion proteins in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Typhoon fla 9000, by GE Healthcare (2 mentions)
Rapalog, by Takara Bio (2 mentions)
Cf568 conjugated secondary antibodies, by Merck Group (2 mentions)
Ptubb3 mc, by Addgene (2 mentions)
Halo ligands jf549, by Promega (2 mentions)
Jf646, by Promega (2 mentions)
4 oh tamoxifen, by Merck Group (2 mentions)
Mouse anti gfp, by Thermo Fisher Scientific (2 mentions)
Mouse anti v5, by Thermo Fisher Scientific (2 mentions)
Rat anti ha, by Merck Group (2 mentions)
Mouse anti flag antibody, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Rabbit anti calnexin, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
4 protocols using rabbit anti halo
1
Affinity Purification of AQP11 Complexes
2
Western Blot Immunostaining Quantification
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Rabbit anti-CBS was purchased from Merck-Millipore. Rabbit anti-Halo was obtained from Promega. Mouse anti-tubulin and rabbit anti-calnexin were from Sigma. Secondary antibodies anti-mouse and anti-rabbit Alexa Fluor 488 or Alexa Fluor 647 were purchased from Invitrogen. Images were acquired using a Typhoon FLA-9000 (GE HealthCare), processed with ImageJ and densitometrically quantified when indicated by ImageJ ±SEM.
3
Antibodies and Fluorescent Labels for Microscopy
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The following primary antibodies were used in this study: mouse anti-GFP 1:2000 dilution (Thermo Fisher Scientific, RRID: AB_221568 ), rabbit anti-GFP 1:2000 (MBL, RRID: AB_591819 ), rabbit anti-Halo 1:1000 (Promega, RRID: AB_713650 ), rat anti-HA 1:500 (Sigma-Aldrich, RRID: AB_390919 ), and mouse anti V5 1:1000 (Invitrogen, RRID: AB_2556564 ). Alexa488-, Alexa568-, and Alexa647-conjugated secondary antibodies were used at 1:400 dilution, obtained from Life Technologies. CF568-conjugated secondary antibodies were used at 1:600 dilution, obtained from Sigma-Aldrich. Molecular biology reagents were obtained from Thermo Fisher Scientific. GFP minicircle for β3-tubulin were custom made at System Biosciences, produced from pTubb3 MC (Addgene #87112, Suzuki et al., 2016 (link)). 4OH-tamoxifen was acquired from Sigma-Aldrich (catalog #H7904), and kept at 20 mm in ethanol in single use aliquots at −20°C. For Halo labeling, Halo Ligands JF549 (Promega, GA1110) and JF646 (Promega, GA1121) were used (Grimm et al., 2015 (link)). Both dyes were dissolved using DMSO to 0.2 mm on arrival. Working concentration was 0.2 μm (1:1000). For FKBP-FRB dimerization, we used Rapalog (TaKaRa, #635057).
4
Multicolor Immunolabeling of Proteins
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The following primary antibodies were used in this study: mouse anti-GFP 1:2000 dilution (Thermo Fisher, RRID: AB_221568), rabbit anti-GFP 1:2000 (MBL, RRID: AB_591819) rabbit anti-Halo 1:1000 (Promega, RRID: AB_713650), rat anti-HA 1:500 (Sigma-Aldrich, RRID: AB_390919) mouse anti V5 1:1000 (Invitrogen, RRID: AB_2556564). Alexa488-, Alexa568-, and Alexa647-conjugated secondary antibodies were used at 1:500 dilution, obtained from Life Technologies. CF568-conjugated secondary antibodies were used at 1:600 dilution, obtained from Sigma-Aldrich. Molecular biology reagents were obtained from Thermo Fisher. GFP minicircle for β3-tubulin were custom made at System Biosciences (Palo Alto, CA, USA), produced from pTubb3 MC (Addgene #87112, Suzuki et al., 2016) (link). 4OHtamoxifen was acquired from Sigma-Aldrich (cat. no H7904), and kept at 20 mM in ethanol in single use aliquots at -20°C. For Halo labeling, Halo Ligands JF549 (Promega, GA1110) and JF646 (Promega, GA1121) were used. Both dyes were dissolved using DMSO to 0.2 mM upon arrival. Working concentration was 0.2 µM (1:1000). For FKBP-FRB dimerization, we used Rapalog (TaKaRa, Shiga, Japan, #635057).
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