The BAF complexes were immuno-purified from nuclear extract using anti-BRG1 or anti-BAF57 (for endogenous BAF) or anti-FLAG antibody (for FLAG-BRG1-containing BAF) and were spotted onto a nitrocellulose membrane (sample amount normalized by cell number). The membrane was allowed to dry at room temperature for 30 minutes. The membrane was rinsed with TBST (150mM NaCl, 20 mM Tris, pH7.4, 0.05% Tween-20), blocked with 5% BSA in TBST, and incubated with Biotin-XX Phalloidin (Biotium) either overnight at 4°C or for two hours at room temperature followed by three washes with TBST. The membrane was incubated with High Sensitivity Streptavidin-HRP (21130, Pierce/Thermo Fisher) for one hour and was washed three times with TBST, followed by detection with ECL reagent.
High sensitivity streptavidin hrp
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
High Sensitivity Streptavidin-HRP is a reagent used in various laboratory techniques involving biotin-streptavidin interactions. It consists of streptavidin, a protein that binds strongly to the vitamin biotin, conjugated to horseradish peroxidase (HRP), an enzyme that can be used for colorimetric or chemiluminescent detection.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Biotin xx phalloidin, by Biotium (2 mentions)
Ab252849, by Abcam (1 mentions)
Ab6556, by Proteintech (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
M180 3, by Abcam (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Irdye 680, by LI COR (1 mentions)
Western lightning plus ecl kit, by PerkinElmer (1 mentions)
Chicken red blood cells, by Rockland Immunochemicals (1 mentions)
Elisa plate, by Corning (1 mentions)
Iblot dry transfer system, by Thermo Fisher Scientific (1 mentions)
Westernbreeze kit, by Thermo Fisher Scientific (1 mentions)
Phospho yap, by Cell Signaling Technology (1 mentions)
Ac 15, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Image station 4000r, by Kodak (1 mentions)
Anti his hrp antibody, by Qiagen (1 mentions)
13 protocols using high sensitivity streptavidin hrp
1
Purification and Detection of BAF Complexes
2
Purification and Detection of BAF Complexes
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The BAF complexes were immuno-purified from nuclear extract using anti-BRG1 or anti-BAF57 (for endogenous BAF) or anti-FLAG antibody (for FLAG-BRG1-containing BAF) and were spotted onto a nitrocellulose membrane (sample amount normalized by cell number). The membrane was allowed to dry at room temperature for 30 minutes. The membrane was rinsed with TBST (150mM NaCl, 20 mM Tris, pH7.4, 0.05% Tween-20), blocked with 5% BSA in TBST, and incubated with Biotin-XX Phalloidin (Biotium) either overnight at 4°C or for two hours at room temperature followed by three washes with TBST. The membrane was incubated with High Sensitivity Streptavidin-HRP (21130, Pierce/Thermo Fisher) for one hour and was washed three times with TBST, followed by detection with ECL reagent.
3
Quantitative Determination of Biotinylated RNA
4
Comprehensive Western Blot Antibody Protocol
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Anti-α-tubulin (Sigma-Aldrich, clone B-5-1-2, 1:1000), anti-METTL18 (Proteintech Group, 25553-1-AP), anti-SETD3 (Abcam, ab174662, 1:1000), anti-RPL3 (Proteintech Group, 66130-1-lg and 11005-1-AP, 1:1000), anti-PES1 (Abcam, ab252849, 1:1000), anti-NMD3 (Abcam, ab170898, 1:1000), anti-HA (Medical & Biological Laboratories [MBL], M180-3, 1:1000), anti-GFP (Abcam, ab6556, 1:1000), anti-RPL17 (Proteintech Group, 14121–1-AP, 1:1000), and anti-β-actin (MBL, M177-3, 1:1000) primary antibodies were used.
ForFigures 1C and 2A , Figure 1—figure supplement 1D , anti-mouse IgG, HRP-Linked Whole Ab Sheep (Cytiva, NA931V, 1:5000) and anti-rabbit IgG, HRP-Linked Whole Ab Donkey (Cytiva, NA934V, 1:5000) secondary antibodies were used. The chemiluminescence was raised with a Western Lightning Plus-ECL Kit (PerkinElmer) according to the manufacturer’s protocol and detected with X-ray film (FUJIFILM, RX-U). For biotinylated proteins, high-sensitivity streptavidin-HRP (Thermo Fisher Scientific, 21130) was used.
To generateFigures 2B and 5C , Figure 3—figure supplement 1D , IRDye680- or IRDye800CW-conjugated secondary antibodies (LI-COR Biosciences, 925-68070/71 and 926-32210/11/19, respectively, 1:10,000) were used. Images were obtained with Odyssey CLx (LI-COR Biosciences).
For
To generate
5
DH1029 Antibody Blocking Assay
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DH1029 blocking by vaccinated mouse sera was performed in ELISA. Briefly, 384-well assay plate were coated with 2 μg/mL PGT145. Then, 0.125 μg/mL of CH848 10.17DT E169K SOSIP trimer were captured for 1 h at room temperature. Next, mouse sera at 1:50 dilution or DH1029 mAb in serial dilution (2-fold dilution starting at 2 ug/mL) were incubated for 1 h. Next, biotinylated DH1029 were added to the plate at 0.05 μg/mL for 1 h and binding were detected by High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific, Cat #21130). Plate development and data acquisition were the same as described above.
6
Quantitative Determination of Biotinylated RNA
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Equal amounts of column-purified, biotinylated RNA were spotted onto 2× SSC equilibrated Hybond-N+ membrane (GE Healthcare), allowing spots to dry in between. The membrane was crosslinked using the auto crosslink function at 254 nm on a UV Stratalinker (Stratagene). The membrane was then blocked in a 10% SDS blocking solution followed by incubation with 1:10,000 dilution of high sensitivity streptavidin-HRP (Thermo Fisher Scientific) in blocking solution. The membrane was washed ×2 in a 1:10 solution of blocking solution and ×2 in Tris-saline buffer. It was then incubated in SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to manufacturer’s instructions and imaged on a ChemiDoc MP imaging system (Bio-Rad) under the chemiluminescence channel. Following imaging, the blot was stained in a 0.04% methylene blue, 0.3 M sodium acetate solution to visualize all RNA as a loading control. The blot was imaged again using the colorimetric channel.
7
DH1029 mAb Blocking Assay
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DH1029 blocking by vaccinated mouse sera was performed in ELISA. Briefly, 384-well assay plate were coated with 2 μg/ml PGT145. Then, 0.125 μg/ml of CH848 10.17DT SOSIP trimer were captured for 1 h at room temperature. Next, mouse sera at 1:50 dilution or DH1029 mAb in serial dilution were incubated for 1 h. Next, biotinylated DH1029 were added to the plate for 1 h and binding were detected by High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific, Cat #21130). Plate development and data acquisition were the same as described above.
8
Cysteine Residue Biotinylation and Detection
9
Quantitative RNA Dot Blot Analysis
10
IgG, IgG1 ELISA and Hemagglutination Inhibition Assay
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