For assessment of protein levels, cells were lysed using RIPA buffer, and 10 μg of total protein was separated and electroblotted as described previously (15 (link)). Protein bands probed with diluted primary antibodies (Table S3 ) were detected using a goat anti-rabbit or mouse IgG-HRP conjugated secondary antibody (200 μg/mL; Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence (Amersham Biosciences).
Goat anti rabbit or mouse igg hrp conjugated secondary antibody
Manufactured by Santa Cruz Biotechnology
Goat anti-rabbit or mouse IgG-HRP conjugated secondary antibody is a laboratory reagent used in immunoassays and Western blotting techniques. It consists of a goat-derived antibody that binds to rabbit or mouse immunoglobulin G (IgG) and is conjugated with the enzyme horseradish peroxidase (HRP). This secondary antibody enables the detection and visualization of target proteins or antigens in samples.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by Cytiva (5 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (2 mentions)
Anti α tubulin antibody, by Cell Signaling Technology (2 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (2 mentions)
Gapdh, by Bio-Techne (1 mentions)
Las 4000 mini camera, by Fujifilm (1 mentions)
Glut1, by Merck Group (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay dye reagent, by Bio-Rad (1 mentions)
Ne pertm nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
7 protocols using goat anti rabbit or mouse igg hrp conjugated secondary antibody
1
Protein Quantification by Western Blot
2
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assessment of protein levels, cells were lysed using RIPA buffer, and 10 µg of total protein was separated and electroblotted as described previously [28 (link)]. Protein bands were detected using a goat anti-rabbit or mouse IgG-HRP conjugated secondary antibody (200 µg/mL; Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence (Amersham Biosciences).
3
Quantifying Glycolytic Enzymes in GIST
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole lysates from GIST tissues were prepared using passive lysis buffer (Promega, Madison, WI) with a protease inhibitor cocktail (Roche, Mannheim, Germany). Total protein lysates (5 μg) were loaded into each lane, size-fractionated by SDS-PAGE and transferred to a nitrocellulose membrane that was blocked with Tris-buffered saline-Tween 20 containing 5% skim milk. Primary antibodies against GLUT1 (1:500; Millipore), HK1 (1:1000; Cell Signaling Technology), PKM2 (1:1000; Cell Signaling Technology), LDHA (1:1000; Cell Signaling Technology), β-actin (1:2000; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:100,000; Trevigen, Gaitherburg, MD) were incubated with the membrane for overnight at 4°C. After washing, membranes were incubated with goat anti-rabbit or mouse IgG-HRP conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), washed, and then developed using western blotting luminol reagent (Santa Cruz Biotechnology). Protein band intensity was analyzed by using a LAS-4000 Mini camera (Fujifilm, Tokyo, Japan).
4
Protein Quantification and Co-Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assessment of protein levels, cells were lysed using RIPA buffer, and 10 µg of total protein was separated and electroblotted as described previously28 (link). Protein bands were detected using a goat anti-rabbit or mouse IgG-HRP conjugated secondary antibody (200 µg/ml; Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence (Amersham Biosciences). Co-immunoprecipitation was performed as previously described39 (link). In brief, rabbit anti-CASZ1 antibody was first incubated overnight with Dynabead M-280 Sheep Anti-Rabbit IgG magnetic beads (ThermoFisher Cat. # 11204D). Whole cell extracts from CTRtetCASZ1b cells treated with Dox for 24 h were incubated for 4 h with the CASZ1 antibody-bound magnetic beads under constant shaking at 4 °C. The co-IP products were eluted by incubating with 1x SDS loading buffer heated to 100 oC for 3 min. Protein bands were detected using a goat anti-rabbit IgG-HRP conjugated secondary antibody (200 µg/ml; Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence (Amersham Biosciences).
5
Quantitative Protein Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assessment of protein levels, cells were lysed using RIPA buffer supplemented with HaltTM protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was determined by using the Bradford dye reagent protein assay (Bio-Rad Laboratories). For nuclear and cytosolic separation, we used the NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Cell lysates in SDS-containing buffer were denatured for 10 minutes, and 10 µg of total protein was resolved by 4–20% SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. Primary antibodies as listed in Supplementary Table 2 were incubated overnight at 4°C in 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 0.02% sodium azide. Secondary antibodies were incubated for 1 hour at room temperature in 5% non-fat dry milk in TBST. Protein bands were detected using a goat anti-rabbit or -mouse IgG-HRP conjugated secondary antibody (200 µg/ml; Santa Cruz Biotechnology) and the SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), visualized by enhanced chemiluminescence (Bio-Rad Laboratories). Quantification was performed with the ImageJ software.
6
Protein Level Assessment and Co-Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assessment of protein levels, cells were lysed 24 hours after transfection using RIPA buffer, and 10µg of total protein was separated and electroblotted as described previously (14 (link)). Anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, catalog #F1804), anti-GAPDH antibody (Santa Cruz Biotechnology, catalog #sc-25778), anti-α-tubulin antibody (Cell Signaling, catalog #2144) were used as primary antibody. Protein bands were detected using a goat anti-mouse or rabbit IgG-HRP conjugated secondary antibody (Santa Cruz Biotechnology, catalog #sc-2030 and sc-2031) and visualized using enhanced chemiluminescence (Amersham Biosciences). Co-immunoprecipitation was performed as previously described (16 ). In brief, HEK293T cells in 10 cm dishes were transiently transfected with empty vector (EV) or FLAG-CASZ1b or mutant constructs for 24 hr and whole cell extracts were incubated with ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich, catalog #M8823) and agitated at 4°C for 4 hr. The co-IP products were eluted by incubating with 2x SDS loading buffer and boiling for 3 min. Rabbit anti-HDAC1, RBAP46/48, MBD3, and PARP1 antibody (Cell Signaling, catalog #2062, #4633, #14540 and #9532), anti- MTA3 antibody (Bethyl, A300-160A) and anti-histone H3 (active motif, catalog #39163) were used as primary antibody.
7
Protein Level Assessment and Co-Immunoprecipitation
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!