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Rabbit α ha

Manufactured by Merck Group
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Rabbit α-HA is a laboratory reagent used in various immunological and biochemical applications. It is an antibody produced in rabbits that specifically binds to the Hemagglutinin (HA) tag, a commonly used protein tag for recombinant protein expression and purification. The core function of Rabbit α-HA is to facilitate the detection and identification of HA-tagged proteins in experimental settings.

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Lab products found in correlation

Mouse α flag m2, by Merck Group (4 mentions) Hrp conjugated secondary antibody, by Merck Group (2 mentions) Fugene hd transfection reagent, by Promega (2 mentions) α rabbit cby1, by Proteintech (2 mentions) α rabbit dzip1, by Proteintech (2 mentions) α mouse actin, by Merck Group (2 mentions) Mouse α ha, by Merck Group (1 mentions) Ecl chemiluminescence system, by Cell Signaling Technology (1 mentions) Xt mops buffer, by Bio-Rad (1 mentions) Triton x 100, by Thermo Fisher Scientific (1 mentions) Flag peptide, by Merck Group (1 mentions) Dithiothreitol (dtt), by Promega (1 mentions) N octyl β d glucopyranoside, by Merck Group (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions) N dodecyl β d maltopyranoside, by Anatrace (1 mentions) Nbd palmitoyl coa, by Avanti Polar Lipids (1 mentions)

5 protocols using rabbit α ha

1

Western Blot Analysis of Protein Interactions

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Protein and IP samples were separated by classic SDS-PAGE, transferred to nitrocellulose membranes and probed with the following commercially available antibodies: rabbit α-Connexin-43 (Cell Signaling), mouse α-Itch (BD Transduction Laboratories), mouse α-HA (Sigma), rabbit α-HA (Sigma), rabbit α-DDB1 (Bethyl Laboratories Inc), rabbit α-Ncoa5 (Bethyl Laboratories Inc), rabbit α-Xpo7 (Proteintech), mouse α-Flag M2 (Sigma), or mouse α-p65 F6 (Santa Cruz Biotechnology). Xpo7 was also analyzed using an antibody which has been described previously (51 (link)) and which was generously provided by Dirk Görlich, Max-Planck Institut f. Biophysikalische Chemie in Göttingen, Germany. After incubation with the appropriate peroxidase-coupled secondary antibodies, signals were visualized with the ECL chemiluminescence system (Cell Signaling).
Landsberg C.D., Megger D.A., Hotter D., Rückborn M.U., Eilbrecht M., Rashidi-Alavijeh J., Howe S., Heinrichs S., Sauter D., Sitek B., Le-Trilling V.T, & Trilling M. (2018). A Mass Spectrometry-Based Profiling of Interactomes of Viral DDB1- and Cullin Ubiquitin Ligase-Binding Proteins Reveals NF-κB Inhibitory Activity of the HIV-2-Encoded Vpx. Frontiers in Immunology, 9, 2978.
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2

Western Blot Analysis of Protein Extracts

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Pooled leaf discs as described above were either homogenised directly in 100 µl Laemmli buffer or were macerated in the Rluc-PCA assay buffer and Laemmli buffer added. The samples were boiled for 5min and cooled on ice. Ten microliters of the homogenate were separated on a 12% 1-mm thick polyacrylamide gel (Criterion™ XT Bis-Tris precast polyacrylamide gel, Biorad, Hercules, CA, USA) in 1× XT-MOPS buffer (Biorad, Hercules, CA, USA). Proteins were transferred to a nitrocellulose membrane and probed with primary and secondary antibodies. Antibodies were diluted in PBS-T 1% (w/v) skimmed milk powder as the following: rabbit α-HA (Sigma-Aldrich, St. Louis, MO, USA), 1:500; swine α-rabbit HRP-conjugate (Dako, Glostrup, Denmark), 1:1700; mouse α-FLAG M2 (Sigma-Aldrich), 1:1000; rabbit α-mouse HRP-conjugate (Dako, Glostrup, Denmark), 1:2000; mouse α-cMyc 9E10 (Sigma-Aldrich), 1:1000, where skimmed milk powder was omitted. Detection was performed with SuperSignal West Dura chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA).
Lund C.H., Bromley J.R., Stenbæk A., Rasmussen R.E., Scheller H.V, & Sakuragi Y. (2014). A reversible Renilla luciferase protein complementation assay for rapid identification of protein–protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus. Journal of Experimental Botany, 66(1), 85-97.
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3

Purification and Labeling of Shh-related Proteins

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n-Dodecyl-β-D-maltopyranoside was purchased from Anatrace (catalog # D310). n-Octyl-β-D-glucopyranoside (OG) was purchased from EMD Millipore (catalog # 494459). Triton X-100 was purchased from Fisher Scientific (catalog # BP151–500). MES was purchased from Fisher Scientific (catalog # BP300–100). DTT was purchased from Promega (catalog # V3151). Anti-FLAG M2 Affinity Gel (catalog # A2220), and 3× FLAG peptide (catalog # F4799) were purchased from Millipore Sigma. Pierce High Capacity Streptavidin Beads were purchased from Thermo Scientific (catalog # 20361). Antibodies were purchased as follows: rabbit α-HA (Sigma, catalog #H6908), mouse α-Shh E-1 (Santa Cruz, catalog # sc-365112), ECL donkey anti-rabbit IgG, horseradish peroxidase linked whole antibody (Cytiva, catalog # NA934), and ECL sheep anti-mouse IgG, horseradish peroxidase linked whole antibody (Cytiva, catalog # NA931). NBD-palmitoyl-CoA, NBD-oleoyl-CoA, and NBD-palmitic acid were obtained from Avanti Polar Lipids. NBD-myristoyl-CoA and NBD-palmitoleoyl-CoA were synthesized by the Memorial Sloan Kettering Organic Synthesis Core Lab. Biotinylated Shh peptides (CGPGRGFGKR-(PEG2)-K(Biotin)-NH2) and (AGPGRGFGKR-(PEG2)-K(Biotin)-NH2) were synthesized by Anaspec and Peptide 2.0.
Schonbrun A.R, & Resh M.D. (2022). Hedgehog acyltransferase catalyzes a random sequential reaction and utilizes multiple fatty acyl-CoA substrates. The Journal of Biological Chemistry, 298(10), 102422.
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4

DZIP1-CBY1 Protein Stability Assay

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HEK293T cells were seeded at 5 × 105/well on six‐well plate. On the next day, cells were co‐transfected with wild‐type DZIP1‐Flag or DZIP1C585W‐Flag and CBY1‐HA constructs using FuGENE HD transfection Reagent (Promega, Cat No: E2311). Cycloheximide was added 48 hours post transfection at concentration of 100 ng/mL. Cells were lysed with RIPA buffer at 0, 4, 8, 16, 32 hours after cycloheximide treatment. Dzip1S14R/+ and wild‐type MEFs were seeded at 2 × 105/well on six‐well plate and treated with cycloheximide at concentration of 100 ng/mL the next day. Cell lysates were harvested using RIPA buffer at 0 and 48 hours post cycloheximide treatment. Immunoblotting was performed as described previously.11 Transfections were performed in triplicate and repeated a minimum of three times. Primary antibodies used for western blot were: α‐mouse Flag M2 (Sigma), α‐rabbit HA (Sigma), α‐rabbit CBY1(Protein tech), α‐rabbit DZIP1 (Protein tech), α‐mouse actin (Millipore). HRP‐conjugated secondary antibodies were purchased from Sigma.
Guo L., Beck T., Fulmer D., Ramos‐Ortiz S., Glover J., Wang C., Moore K., Gensemer C., Morningstar J., Moore R., Schott J., Le Tourneau T., Koren N, & Norris R.A. (2021). DZIP1 regulates mammalian cardiac valve development through a Cby1‐β‐catenin mechanism. Developmental Dynamics, 250(10), 1432-1449.
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5

DZIP1 Protein Stability Assay

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HEK293T cells were seeded at 5 × 105/well on six-well plate. On the next day, cells were co-transfected with wild-type DZIP1-Flag or DZIP1C585W-Flag and CBY1-HA constructs using FuGENE HD transfection Reagent (Promega, Cat No: E2311). Cycloheximide was added 48 hours post transfection at concentration of 100 ng/mL. Cells were lysed with RIPA buffer at 0, 4, 8, 16, 32 hours after cycloheximide treatment. Dzip1S14R/+ and wild-type MEFs were seeded at 2 × 105/well on six-well plate and treated with cycloheximide at concentration of 100 ng/mL the next day. Cell lysates were harvested using RIPA buffer at 0 and 48 hours post cycloheximide treatment. Immunoblotting was performed as described previously.11 (link) Transfections were performed in triplicate and repeated a minimum of three times. Primary antibodies used for western blot were: α-mouse Flag M2 (Sigma), α-rabbit HA (Sigma), α-rabbit CBY1(Protein tech), α-rabbit DZIP1 (Protein tech), α-mouse actin (Millipore). HRP-conjugated secondary antibodies were purchased from Sigma.
Guo L., Beck T., Fulmer D., Ramos‐Ortiz S., Glover J., Wang C., Moore K., Gensemer C., Morningstar J., Moore R., Schott J., Le Tourneau T., Koren N, & Norris R.A. (2021). DZIP1 regulates mammalian cardiac valve development through a Cby1‐β‐catenin mechanism. Developmental Dynamics, 250(10), 1432-1449.
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