Protein isolated from whole cell lysate was separated on 4–20% acrylamide gel (Mini-Protean GTX, BioRad). Protein was transferred to PVDF membranes and blocked with 10% milk for 1 h. Gels were stained post-transfer using Coomassie stain to visualize loaded protein levels. Blots were probed with anti-GRB2 antibodies (dilution of 1:5,000; Abcam, Ab32037), anti-phosho-CRKL antibodies (dilution of 1:1000; Cell Signaling Technology Inc., 3181S), and anti-phospho-c-ABL1 antibodies (dilution of 1:1000; Cell Signaling Technology Inc., 2868S). Anti-GAPDH antibodies (dilution of 1:15,000; Abcam, Ab181602) were used as a loading control. Goat anti-rabbit IgG coupled to HRP secondary antibodies were used at 1:10,000 dilution (Abcam). Blots were stained with Ponceau stain to confirm loading amounts. ImageJ was used to quantitate pixel density.
Ab32037
Manufactured by Abcam
Sourced in United States
Ab32037 is a laboratory equipment product. It is a device used for a specific function in the laboratory setting. No further details about its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab181602, by Abcam (1 mentions)
Ab125025, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Ab23914, by Abcam (1 mentions)
Ab76956, by Abcam (1 mentions)
Ab278538, by Abcam (1 mentions)
Dab solution, by Merck Group (1 mentions)
Rm2235, by Leica camera (1 mentions)
D8001, by Merck Group (1 mentions)
Goat anti rabbit igg secondary antibody, by Merck Group (1 mentions)
Ab4074, by Abcam (1 mentions)
Abs20001, by Absin (1 mentions)
Abs20002, by Absin (1 mentions)
Supersignal west pico trial kit, by Thermo Fisher Scientific (1 mentions)
0.45 mm nitrocellulose membrane, by Merck Group (1 mentions)
Mab8131, by Merck Group (1 mentions)
6 protocols using ab32037
1
Protein Expression Analysis by Western Blot
2
Western Blotting for GRB2 and ROCK2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3
Western Blot Analysis of HCC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We lysed the HCC cells and patient tissues using cold RIPA buffer containing protease inhibitors (1:100) and protein inhibitors (1:100). Then, the protein concentration in all samples was estimated using the BCA assay. Equal amounts of protein samples were separated on a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membranes were blocked using 5% skimmed milk for 1 h. Then, the membranes were incubated overnight at 4oC with primary antibodies against GRB2 (1:1000; ab32037, Abcam), E-cadherin (1:1000; #3195, CST), N-cadherin (1:1000; #13116, CST), Vimentin (1:1000; #5741, CST) and β-actin (1:10000; ab6276, Abcam). Then, the membranes were incubated with the secondary HRP-conjugated antibody (KPL, USA). Then, the blots were developed using ECL (EMD Millipore, MA, USA). The ImageJ software was used to determine the relative expressions of different proteins using β-actin as the internal control.
4
Immunohistochemical Analysis of Spinal Ligament Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Interlaminar ligamentum flavum, interspinous ligament, or supraspinal ligament tissue was obtained from AS patients and traumatic injury patients who had spinal fractures when undergoing spinal surgery as controls. The tissues were fixed in 4% paraformaldehyde for 4–6 h and then embedded in paraffin. The paraformaldehyde-fixed paraffin-embedded sections were cut into 5 μm using a Leica RM2235. Paraffin-embedded sections were first deparaffinized twice in a series of 100% xylene, rehydrated in a series of graded ethanol (100%, 100%, 95%, and 80%), and then washed briefly in distilled water and PBS, respectively. Antigen thermal repair was performed with 0.01 M citrate buffer (pH6.0) or EDTA buffer (pH9.0) at high pressure, washed in PBS, sections were treated with 3% hydrogen peroxide-methanol for 10 min and blocked with normal goat serum for 30 min. Then, the sections were incubated overnight with PDGFB (1:100, Abcam, catalog: ab23914); GRB2 (1:50, Abcam, catalog: ab32037); P-ERK (1:100, Abcam, catalog: ab278538); RUNX2 (1:50, Abcam, catalog: ab76956) antibody at 4 °C. Goat anti-rabbit IgG secondary antibody (JACKSON, catalog: 111–035-003) was incubated and DAB solution (Sigma, catalog: D8001) was used for color development. All images were obtained using an Open-field slice scanner (NanoZoomer S210).
5
Immunohistochemistry for Grb2 and α-tubulin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed on formalin-fixed paraffin-embedded tissue sections (20 μm), as previously described (Coleman and Stanley, 1994 (link)), by using antibodies against the following proteins at manufacturer-recommended dilutions: Grb2 and α-tubulin (Abcam, # ab32037 and ab4074, respectively. ImageJ software was used for the calculation of the intensity correlation quotient.
6
Western Blot Analysis of Spleen Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from the spleen and quantified using the BCA method. Equal amounts of protein per sample were resolved with 10% SDS-PAGE and transferred to a 0.45-mm nitrocellulose membrane (EMD Millipore, Billerica, MA, USA) for 1 h. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated overnight with the specific primary antibodies [diluted 1:1,000 in 5% skim milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.15% Tween-20)] against GRB2 (ab32037; Abcam), β-Actin (bsm-33036M; Bioss Bioscience), and IE2 (MAB8131; Millipore). The membranes were washed and separately placed in secondary antibodies. The membranes of GRB2 and β-actin were placed in anti-mouse IgG-HRP (abs20001; Absin Bioscience), and the membrane of IE2 was placed in anti-rabbit IgG-HRP (abs20002; Absin Bioscience) (both diluted 1:2,000 in 5% skim milk in TBST) for 2 h at room temperature. After membranes were washed three times with TBST at room temperature, positive bands were developed using the SuperSignal West Pico Trial kit (Thermo Fisher Scientific, Inc.) and detected with the VilberLourmat imaging system (VilberLourmat, Marne-la-Vallée, France). (Beckman Coulter). The results were analyzed with the software FlowJo Version 10 (TreeStar).
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!