HeLa 11ht cells were seeded at a concentration of 1 × 105 cells per well of a six-well plate or 8 × 105 cells per 10 cm dish the day before transfection. Transfection was performed using polyethylenimine (Polysciences, Inc.) in a 2:1 (w/w) ratio to the added DNA amount. Plasmid DNA (3 μg for six-well and 6 μg for 10 cm), polyethylenimine and DMEM (without serum) were mixed in a total volume of 500 μl, incubated for 5 min at RT and added to the cells. The media of the cells was changed directly before transfection. The day after transfection, biotin (Sigma) was added to the medium (50 μM) and the cells were induced with 200 ng ml−1 doxycycline (Sigma). In addition, for FRB/FKBP constructs, rapamycin (Invivogen) was added to 100 nM. Lysates were prepared 24 h after induction as follow: cells were washed once with PBS and scraped with 100 μl lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT and complete protease inhibitor (Roche)) followed by a centrifugation step at 14,000g for 10 min at 4 °C. Protein amounts were determined with a Bradford assay (Expedeon) and equal protein amounts were loaded on SDS–PAGE gels and analysed by western blot.
Bradford assay
Manufactured by Abcam
Sourced in United Kingdom
The Bradford assay is a colorimetric protein quantification method. It measures the absorbance of a dye-protein complex formed when a protein sample is mixed with the Bradford reagent. The absorbance is proportional to the protein concentration in the sample, allowing for the quantification of total protein levels.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Lightning plus ecl enhanced chemiluminescence substrate, by PerkinElmer (2 mentions)
Pho ss ro, by Merck Group (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rapamycin, by InvivoGen (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Mab002, by R&D Systems (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by GE Healthcare (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
Gapdh antibody, by Abcam (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
G box imaging system, by Syngene (1 mentions)
Nuclear extraction kit, by Abcam (1 mentions)
12 protocols using bradford assay
1
Inducible Biotinylated Protein Expression
2
Co-immunoprecipitation of Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For co-immunoprecipitations (co-IP), cells grown in 10 cm dishes were lysed in ice-cold co-IP buffer (50 mM Tris-HCl, pH 7.4, 1% Triton-X-100, 25 mM Hepes, 150 mM NaCl, 0.2% Sodium deoxycholate, 5 mM MgCl2 and protease inhibitor cocktail Roche). Cell lysates were incubated for 60 min on a rotary wheel at 4°C. After centrifugation for 15 min at 13200 rpm, the supernatant was transferred into a new 1.5 ml tube and protein concentration measured by a Bradford Assay (Expedeon, Swavesey, UK). Equal amounts of cell lysates (200 μg) were pre-cleared for 1 h at 4°C on a rotary wheel. Pre-cleared lysates were incubated with 3 μg of anti-myc (9E10) antibody or 3 μg of control mouse IgG (mAB002, R&D Systems, Minneapolis, USA) and incubated over-night at 4°C on a rotary wheel. Dynabeads Protein G (Life Technologies) were added to the immunocomplexes and incubated for 1 h at 4°C on a rotary wheel. After 3 washes with co-IP buffer, complexes were eluted in denaturing SDS sample buffer, resolved by SDS-PAGE and analyzed by immunoblotting
3
Western Blot Analysis of ADAR1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from cells using the RIPA Lysis Buffer System (Santa Cruz Biotechnology) and the protein concentration was determined via the Bradford assay (Expedeon, Swavesey, UK). Samples (50–90 μg protein) were mixed with 5x Laemmli buffer and heated at 95°C for 5 min for denaturation. Proteins were fractionated by SDS-PAGE and transferred to Polyvinylidene difluoride membranes (Immobilon-P, Millipore, Darmstad, Germany). Blocking was performed with 5% bovine serum albumin (GE Healthcare) for 10 min. Primary antibodies were added overnight at 4°C. The antibody against ADAR1 (Santa Cruz Biotechnology) was diluted 1:1000 and the GAPDH antibody (Abcam, Cambridge, UK) was diluted 1:10000. After extensive washing, membranes were incubated with secondary HRP-conjugated antibodies, anti-mouse or anti-rabbit (Thermo Fisher Scientific), diluted 1:4000. Membranes were extensively washed before detection. Pierce ECL western blotting substrate (Thermo Fisher Scientific) or Super Signal West FEMTO (Thermo Fisher Scientific) was used for detection by means of chemiluminescence using LAS-4000 mini camera system (Fujifilm Life Science Systems, Japan).
4
Brentuximab-MMAE ADC Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brentuximab-MMAE ADCs were prepared as previously described (Godwin, 2017 ; Godwin et al., 2017 ). Briefly, brentuximab (in 20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 20 mM EDTA) was reduced at 5 mg/ml antibody concentration with TCEP (1.5 equivalents per disulfide) for 1 h at 40°C. Reduced antibody solutions were cooled to 22°C and diluted to 4.21 mg/ml with 20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 20 mM EDTA. Conjugation reagents (1a-f , 2a-b and 3a-b ) in MeCN were added (5% v/v MeCN, 1.4 equivalents per disulfide) to the reduced antibody. Conjugation reactions were incubated for16-20 h and subsequently quenched by the addition of 50 mM N-acetyl-l -cysteine (3 mM final concentration). The crude reactions were then purified by hydrophobic interaction chromatography (HIC) using ToyoPearl Phenyl-650S columns, as described previously (Bird et al., 2020 (link)). Fractions containing DAR 4 ADC were pooled and buffer exchanged into PBS. Antibody concentration was determined by Bradford assay (Expedeon). DAR 4 was determined by HIC-HPLC, a more quantitative characterisation than SDS-PAGE or UV-vis (see Bird et al., 2020 (link))
5
Validating Bait-BirA Protein Secretion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the secretion of bait-BirA proteins, supernatants of cultures expressing fusion proteins were collected, centrifuged to remove cell debris, and protein content was concentrated using Amicon Ultra 4 mL 10 KD filter unit (MilliporeSigma) and quantified using Bradford assay (Expedeon). 20 ug of total protein was loaded on SDS-PAGE gel for electrophoresis and resolved proteins were transblotted to nitrocellulose membrane using Trans-Blot Turbo Transfer System from Bio-RAD. The membrane was blocked with 5% skim milk in TBST and probed with HRP-conjugated anti-flag mouse monoclonal antibody (Thermofisher) diluted at 1:10000 in the blocking buffer. The membrane was washed, and Clarity Western ECL Substrate was added. Proteins' bonds were visualized using G:Box Gel Image Analysis Systems (SYNGENE). For staining of intracellular biotinylated proteins, cells were grown in complete medium supplemented with 50 μM biotin, lysed by RIPA buffer, and protein content was quantified using Bradford assay. 20 ug of total protein was loaded and resolved and transblotted as described earlier. The membrane was blocked by 3% BSA in TBST and probed with HRP-conjugated streptavidin diluted in blocking buffer at 1:2000 ratio. For visualizing the proteins' bands, the same Clarity Western ECL Substrate was used.
6
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were homogenized in RIPA buffer containing protease inhibitor cocktail. Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific. Cat. 23225) or Bradford Assay (Cat. ab119216, Abcam). The blot was performed using 15 to 20 µg of protein according to previously described methods [26 (link), 27 (link)]. Primary and secondary antibodies used are detailed in Tables 1 and 2 . Membranes were visualized with enhanced chemiluminescence (Clarity Western ECL Blotting Substrates; Bio-Rad Laboratories) and bands detected using Syngene G-Box imaging system (Syngene). Western Blot analyses were performed using ImageJ software and densitometry normalized to loading control GAPDH or Rab11.
7
TFEB Nuclear Translocation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MCEC WT and KO cells were treated in assay media for 16 hours. Nuclear and cytosol extraction was carried out using a Nuclear Extraction kit (#ab113474; Abcam, Cambridge, UK) following the manufacturer's instructions. Protein concentration was determined using the Bradford assay (#ab102535; Abcam). Equal amounts of cytosolic fractions (approximately 1.5% of total cytosol extract), and equal amounts of nuclear fractions (approximately 15% of the total extract) were loaded to perform Wes immunoassay using antibodies against transcription factor EB (TFEB). Lamin A/C and GAPDH were used as nuclear and cytoplasm references, respectively. Antibody information and dilutions are indicated in Table 1 .
8
Protein Expression Analysis of Prostate Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prostatic organoids and PCa cell lines were lysed in RIPA buffer, supplemented with protease (05892970001; Sigma‐Aldrich) and phosphatase PhoSTOP (PHO SS‐RO; Sigma‐Aldrich) inhibitors. Protein concentrations were determined using Bradford assay (Abcam; ab119216). Equal amounts of proteins were resolved on SDS–PAGE, and transferred onto nitrocellulose membranes using Trans‐blot turbo transfer system (Bio‐Rad).
Membranes were incubated with anti‐CC3 (CST 9664), anti‐cleaved PARP (CST 9544), anti‐HIF1A (CST, 36169), anti‐histone H3 (CST, 4499S), and anti‐ß‐actin (SCBT SC‐47778 or Sigma‐Aldrich A5441) primary antibodies diluted at 1:1,000. Membranes were then incubated with anti‐Mouse IgG (CST 7076S) or anti‐Rabbit IgG (CST 7074S) HRP‐linked antibodies diluted at 1:5,000. Signals were developed using Lightning Plus‐ECL Enhanced Chemiluminescence Substrate (Perkin Elmer; ref: NEL104001EA) and detected using an Amersham™ Imager 600 (GE Healthcare).
Membranes were incubated with anti‐CC3 (CST 9664), anti‐cleaved PARP (CST 9544), anti‐HIF1A (CST, 36169), anti‐histone H3 (CST, 4499S), and anti‐ß‐actin (SCBT SC‐47778 or Sigma‐Aldrich A5441) primary antibodies diluted at 1:1,000. Membranes were then incubated with anti‐Mouse IgG (CST 7076S) or anti‐Rabbit IgG (CST 7074S) HRP‐linked antibodies diluted at 1:5,000. Signals were developed using Lightning Plus‐ECL Enhanced Chemiluminescence Substrate (Perkin Elmer; ref: NEL104001EA) and detected using an Amersham™ Imager 600 (GE Healthcare).
9
Protein Expression Analysis of hNPCs-OE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After collection of the conditioned medium, hNPCs-OE were lysed with RIPA buffer (1X PBS, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, 0.24 mg/ml AEBSF, 8 mg/ml aprotinin, 10 mg/ml leupeptin, 4 mg/ml pepstatin, 5 mM benzamidine, 20 mM glycerophosphate, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA and 1 mM EGTA; Sigma-Aldrich). The total protein content was quantified using a Bradford assay (Abcam, MA, USA). Proteins were separated in an SDS-PAGE system (4 to 12%) and transferred to nitrocellulose paper. The membrane was blocked with 5% of non-fat milk, and proteins were identified with primary antibodies against mouse anti-67kDa laminin receptor (1:3000, Abcam), rabbit anti-Sox2 (1:1000, Sigma-Aldrich), rabbit anti-MASH1 (1:1500, Santa Cruz Immunoresearch, TX, USA), rabbit anti-tubulin beta-III (1:1000, Promega). Beta-actin was used as a loading control (1:2000; Abcam) (Supplementary Figure 1
10
Quantifying RPE Secretome Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Supernatants of primary murine RPE cells treated with C5a or TGF-β2 for different lengths of time was used for determine the concentration of C5a, C5, TGF-β1, TGF-β2 and VEGF. ELISA kits for C5a, C5, TGF-β1, TGF-β2 and VEGF (R&D Systems), IL-6 and TNF-α (Thermo Fisher Scientific-Invitrogen) (Table 2 ) were used according to the manufacturers’ instructions. The concentration obtained in pg/mL was then normalized by the total protein concentration of each sample measured with Bradford assay (Cat. ab119216, Abcam).
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!