For analyzing cells encapsulated in hydrogels, the hydrogels were degraded by incubation with 1 mg/mL collagenase I (Worthington) solution for 40 min and the released cells were pelleted by centrifugation. Cells were homogenized in NP-40 lysis buffer supplemented with protease inhibitor (Sigma-Aldrich P2714) and phosphatase inhibitor cocktails (Cell Signaling Technology 5870S). The lysate was centrifuged at 10 000g for 10 min. The supernatant was stored at –80 °C until further analysis and the pellet was resuspended in RIPA buffer. Equal total protein amount samples, as measured by micro BCA, were loaded on SDS-PAGE gels. The separated proteins were transferred onto nitrocellulose membranes, blocked using 5% bovine serum albumin dissolved in tris-buffered saline with 0.1% Tween-20 (BSA-TBST) at RT for 1 h, incubated with primary antibody at 4 °C overnight, washed with TBST three times for 10 min each, incubated with 1:5000 v/v horseradish peroxidase-conjugated secondary antibody (Bio-Rad) at RT for 1 h, washed three times for 10 min each, incubated with the substrate ECL (Denville Scientific) or ECL Prime (GE) and then exposed to an X-ray film (Denville Scientific). The films were scanned at 600 dpi resolution and obtained bands were quantified by densitometry analysis in ImageJ (NIH). Information on antibodies used is provided in Table S2 .
Enhanced chemi luminescence (ecl)
Manufactured by Thomas Scientific
ECL (Enhanced Chemiluminescence) is a laboratory equipment that generates and detects chemiluminescent signals. It is used in various applications, such as Western blotting, where it can be employed to visualize and quantify specific proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
X ray film, by Thomas Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Collagenase 1, by Worthington (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Nxa931, by GE Healthcare (1 mentions)
Phospho atm ser1981, by Cell Signaling Technology (1 mentions)
Pp h6705 00, by R&D Systems (1 mentions)
β actin, by Merck Group (1 mentions)
2 protocols using enhanced chemi luminescence (ecl)
1
Hydrogel-Encapsulated Cell Protein Analysis
2
Immunoblotting of DNA Damage Response
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Following polyacrylamide gel electrophoresis and protein transfer, nitrocellulose membranes were blocked in 5% nonfat dry milk*** dissolved in Tris-Buffered Saline with Tween-20 (TBST), then probed overnight at 4°C with the following primary antibodies in TBST: ERRbeta #PP-H6707-00 (cl.07) 1:250 - 1:500 and #PP-H6705-00 (cl.05) 1:500 – 1:750 (R&D Systems, Minneapolis, MN); PARP #9542 1:1000, phospho Ser139 (γ) histone H2A. X #9718 1:1000, ***total histone H2A. X #2595 1:1000, phospho Ser1981 ATM #5883 1:500, phospho Ser428 ATR #2853 1:500, phospho Thr68 Chk2 #2197 1:500, phospho Ser345 Chk1 #2348 1:500, phospho Ser10 histone H3 #3377 1:1000, total histone H3 #9715 1:1000, phospho Thr180/Tyr182 p38 MAPK #9216 1:250, p38 #9212 1:500, vinculin #13901 1:1000 (Cell Signaling, Danvers, MA). All membranes were re-probed with β-actin (Sigma #A5316 1:5000 – 1:10,000) as a loading control for ≥1 h at room temperature or overnight at 4°C. Horseradish peroxidase enzyme-conjugated anti-mouse or anti-rabbit whole immunoglobulin (IgG) secondary antibodies (GE #NXA931 and #NA934V, respectively, Buckinghamshire, U.K.) were used at 1:5000 for ≥1 h at room temperature, followed by enhanced chemiluminescence (ECL, Denville Scientific, Holliston, MA) as in [27 (link)]. ***Membranes to be probed for total histone H2A. X were blocked in 5% horse serum in TBST rather than milk.
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