Bladder cancer cells were collected and lysed in 50 μL cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitors (Sigma-Aldrich). The protein concentration was quantified using a BCA Protein Kit (Thermo Fisher Scientific, Rockford, IL, USA). The cell lysates were separated by 10% SDS-PAGE and the proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA), blocked with TBS/T containing 5% BSA, and then incubated with primary antibodies against E-cadherin, vimentin, Twist-1, Zeb-1, snail, or eIF5A2 (Abcam, Cambridge, MA, USA) at 4°C overnight. The membranes were washed three times with TBS/T and then incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. The protein bands were detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA) and visualized by autoradiography (Kodak, Rochester, NY, USA).
50 μl cell lysis buffer
Manufactured by Cell Signaling Technology
Sourced in United States
The 50 μL cell lysis buffer is a solution designed for the extraction and solubilization of proteins from cell samples. It provides the necessary components to efficiently lyse cells and release their intracellular contents, including proteins, for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Vimentin, by Abcam (3 mentions)
E cadherin, by Abcam (3 mentions)
Chemiluminescence, by GE Healthcare (2 mentions)
Autoradiography, by Kodak (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Abcam (2 mentions)
Eif5a2, by Abcam (2 mentions)
Twist1, by Abcam (1 mentions)
Anti p53 anti bodies, by Abcam (1 mentions)
Anti topbp1, by Abcam (1 mentions)
Protease inhibitor, by Abcam (1 mentions)
Twist, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
5 protocols using 50 μl cell lysis buffer
1
Immunoblotting Analysis of EMT Markers
2
TopBP1 and p53 Immunoprecipitation
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Cells were harvested and lysed in 50 μL cell lysis buffer (Cell Signaling) after treatment with doxorubicin for 48 hours. An aliquot of the cell lysates was lysed with SDS lysis buffer, and the rest of the cell lysates were incubated with appropriate antibodies or beads for 24 hours at 4°C. Immunoprecipitates were fractionated by SDS-PAGE and electrotransferred to the polyvinylidenedifluoride membranes (Merck Millipore). The specific signals were detected with anti-TopBP1 or anti-p53 antibodies (Abcam). Protein expression was detected using chemiluminescence (GE Healthcare Bio-Sciences Corp.).
3
Western Blot Analysis of TopBP1 and p53
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Cells were lysed in 50 μL cell lysis buffer (Cell Signaling, Danvers, MA, USA). The protein concentration was quantified using the BCA Protein Kit (Thermo, Rockford, IL, USA). Cell lysates were separated by 10% SDS-PAGE and proteins were transferred to polyvinylidenedifluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were then incubated with anti-TopBP1 or anti-p53 antibodies (Abcam, Cambridge, MA, USA) at 4°C overnight. The membranes were washed three times with TBS/T and then incubated with the appropriate HRP-conjugated secondary antibodies (Abcam) for 1 hour at room temperature. Protein expression was detected by chemiluminescence (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).
4
Western Blot Analysis of EMT Markers
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Tumor cells were lysed in 50 μL cell lysis buffer (Cell Signaling, Danvers, MA, USA) containing protease inhibitors (Sigma, USA). Whole cell lysates were prepared and fractioned were separated by 10 % SDS-PAGE and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then incubated with primary antibodies (E-cadherin, Vimentin or eIF5A2, diluted 1:1000; Abcam, Cambridge, USA) at 4 °C overnight. The membranes were washed three times with TBST and then incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein expression was detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
5
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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OSCC cells were lysed in 50 μl cell lysis buffer (Cell signaling, Danvers, MA) containing protease inhibitors (BioVision) according to the manufacturer's instructions. The cell lysates were quantified using the BCA Protein Kit (Thermo Fisher Scientific, Rockford, IL). 10 % SDS-PAGE was used to separated cell lysates and proteins were transferred to PVDF membranes (Millipore, Billerica, MA), blocked with TBS/T containing 5 % BSA for 2 h on ice. The membranes were incubated with indicated primary antibodies (Vimentin, E-cadherin, eIF5A-2, Twist or GAPDH, dilution 1:1000, Abcam, Cambridge, MA) at 4 ℃ overnight. After washing three times with TBS/T, the membranes were incubated with the specific HRP-conjugated secondary antibodies (Abcam, Cambridge, MA, dilution 1:2000) at room temperature for 1 h. The protein expression was detected by chemiluminescence (GE Healthcare, Piscataway, NJ) and imagined by autoradiography (Kodak, Rochester, NY). The quantifications of protein were done by estimation of protein bands densities (TIFF images) using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD) and protein expression was standardized to GAPDH.
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