Total protein was obtained from transfected SiHa or CaSki cells which were lysed by radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Jiangsu, China). Subsequently, protein was quantified by using the BCA assay kit (Beyotime). The dodecyl sulfate, sodium salt (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed on proteins from lysates which were then moved to polyvinylidene fluoride membranes (PVDF; Millipore, Bedford, MA, USA). Membranes were cultured with primary antibodies overnight at 4 °C, and antibodies used were as follows: anti-Bcl-2 (ab32124, Abcam, Cambridge, USA), anti-Bax (ab32503, Abcam), anti-MMP2 (ab215986, Abcam), anti-MMP9 (ab219372, Abcam), anti-PBX1 (ab97994, Abcam) and anti-GAPDH (ab97994, Abcam). Then, secondary antibodies were added for cultivation for 1 h in dark room. Finally, the proteins were detected by an Enhanced Chemiluminescence Detection Kit (Millipore, Bedford, MA, USA).
Ab97994
Manufactured by Abcam
Sourced in United States
Ab97994 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but no further details about its core function can be provided in a purely factual and unbiased manner without the risk of extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab23981, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab219372, by Abcam (1 mentions)
Ab215986, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Wet transfer blotting system, by Bio-Rad (1 mentions)
Nitrocellulose filter membrane, by Pall Corporation (1 mentions)
Chemiluminescence detection system, by Merck Group (1 mentions)
Ab36861, by Abcam (1 mentions)
Ab83259, by Abcam (1 mentions)
Ab94744, by Abcam (1 mentions)
No c510003, by Sangon (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ab9484, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab4441, by Abcam (1 mentions)
5 protocols using ab97994
1
Protein Expression Analysis in Cervical Cancer
2
Protein Expression Analysis in Bone Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using a commercial kit (No.C510003, Sangon Biotech, China) according to the manufacturer's instructions. Primary antibodies rabbit anti-RUNX2 (ab23981, Abcam), rabbit anti-OSX (ab94744, Abcam), rabbit anti-ACAN (ab36861, Abcam), rabbit anti-OCN (23418-1-AP, ProteinTech, Wuhan, China), rabbit anti-ALP (ab83259, Abcam) and rabbit anti-PBX1 (ab97994, Abcam) were used (all at 1:1,000 dilution). The protein samples were separated on 10% SDS-PAGE gels and subsequently transferred to nitrocellulose filter membranes (Pall Corp.,Washington, NY) using the wet transfer blotting system (BioRad, Hercules, CA). After incubation, secondary antibody goat anti-rabbit-HRP (Pierce, USA) was used at 1:2,000 dilution. The proteins were then detected by chemiluminescence detection system (Millipore, USA). Anti-GAPDH was used as an endogenous control.
3
Quantitative Analysis of PBX1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysis buffer (Beyotime Biotechnol, China) including 1 mM phenylmethyl-sulfonylfluoride (PMSF) was adopted, with the intention of treating the harvest cells for 20 min on ices. Centrifuge the cell lysates at 12,000 rpm for 15 min. A BCA kit (Cat# 23,225, Pierce, USA) was employed, with the intention of measuring the supernatant protein concentration. The protein was separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred with polyvinyl difluoride (PVDF) membranes. Five percent nonfat milk was applied, with the objective to blocking membranes at normal temperature for 2 h. Then, first antibodies of anti-PBX1 (1:1000, Ab97994, Abcam) and anti-GAPDH (1:1000, Ab9484, Abcam) were added to the membranes at the temperature of 4° during the night. We adopted TBS-T to rinse those membranes for three times. HRP-conjugated secondary antibody (1:2000, Ab205718, Abcam) was fostered using those membranes at normal temperature for 1 h. Protein bands were seen by means of one enhanced chemiluminescence (ECL) kit (Millipore, MA, USA). The protein band intensity was measured via ImageJ software. The internal reference was GAPDH.
4
Chromatin Immunoprecipitation and Transcriptional Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation was performed using EZ-Magna ChIP A/G Kit (Millipore, Billerica, MA, USA) following the manufacturer's instructions. Briefly, PLL/OPLL cells transfected with miR-181a-5p agomirs or antagomirs were fixed using formaldehyde with final concentration of 1% to perform cross-linking after incubation for 20 minutes at room temperature, glycine was added to terminate the process at final concentration of 0.2M. Cells were harvested and lysed by lysis buffer supplemented with protease inhibitors, and the lysates were collected for sonification (4 cycles of 25s on, 59 s off, pulse at a power output of 20%) at 4°C. For immunoprecipitation, the samples were incubated with rabbit anti-PBX1 (ab97994, Abcam), anti-RUNX2 (ab23981, Abcam), anti-HOXA10 (ab175026, Abcam), anti-H3K9ac (ab4441, Abcam), anti-H2K9me2 (ab32521, Abcam), anti-RNAPol II (ab5095, Abcam) or a negative rabbit IgG antibody overnight at 4°C, which was followed by incubation with protein A/G agarose resin. The purified immunoprecipitated DNA was further quantified by real-time PCR analysis. The information for all primers is listed in Supplementary Data 2.
5
Immunohistochemical Analysis of PBX1 and OCN
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The specimens were decalcified in 10% ethylene diamine tetraacetic acid (EDTA, pH 7.4) for 1 month, followed by dehydration and embedding in paraffin. Sections (5 μm) were cut and stained with hematoxylin and eosin (H&E). For immunohistochemistry analysis, sections were blocked with 3% BSA for 30 min and then incubated with primary antibody against PBX1 (ab97994, Abcam, USA) and OCN (23418-1-AP, ProteinTech, Wuhan, China) were used at 1:100 dilutions. Antigen retrieval was performed in 95°C citrate buffer (pH 6) for 10 min, then sections incubated with primary antibody at 4°C overnight. After processed by the ABC detection kit (Vector Laboratories, Burlingame, CA), sections were visualized under an Olympus BX51 light microscope equipped with Olympus DP70 camera (Olympus Co., Tokyo, Japan) and quantified using the ImageJ software (US National Institutes of Health).
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!