For protein analysis, we performed eight laser impacts per rat eye. The experiment was repeated twice independently. The dissected RPE-choroidal samples were processed using standard methods23 (link) for western blotting. Briefly, equal amounts of protein (20 µg) were separated on Novex® 4–20% Tris-Glycine gel (Thermo Fisher Scientific), transferred to nitrocellulose, and the blots were incubated with rabbit anti-DCN (ab175404, 1:1000, Abcam, Cambridge, UK) at 4 °C overnight. The membranes were then washed, incubated with HRP goat anti-rabbit IgG antibody (PI-1000, 1:5000, vector) for 1 h at room temperature, and developed using ECL Plus western blotting detection reagents (GE healthcare, Orsay, France). Scans of blots were provided as Supplementary Fig. 10 . The goat anti-Actin (sc1616, 1:2500, Santa Cruz Biotechnology, Heidelberg, Germany) was used as internal control.
Goat anti actin sc 1616
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Goat anti-actin (sc-1616) is a primary antibody product from Santa Cruz Biotechnology. It is an affinity-purified antibody raised against a peptide mapping at the C-terminus of actin of human origin.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Irdye conjugated antibody, by LI COR (2 mentions)
Sds page gel, by Thermo Fisher Scientific (2 mentions)
Lds electrophoresis loading buffer, by Thermo Fisher Scientific (2 mentions)
Abs45, by Merck Group (2 mentions)
660 nm protein assay reagent, by Thermo Fisher Scientific (2 mentions)
Odyssey imager, by LI COR (2 mentions)
M per lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab175404, by Abcam (1 mentions)
Novex 4 20 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Mouse anti gli1 l42b10, by Cell Signaling Technology (1 mentions)
Rabbit anti parp 9542, by Cell Signaling Technology (1 mentions)
Rabbit anti hdac1 h3284, by Merck Group (1 mentions)
Ab24170, by Abcam (1 mentions)
Ab137029, by Abcam (1 mentions)
Rabbit anti eea1, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Rpn2209, by GE Healthcare (1 mentions)
8 protocols using goat anti actin sc 1616
1
Quantitative Protein Analysis in Rat Eyes
2
Immunoblot Analysis of Signaling Proteins
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Tissues were lysed in a solution containing RIPA buffer (50 mM Tris-HCl at pH 7.6, 150 mM NaCl, 0.5% sodium deoxycholic, 5 mM EDTA, 0.1% SDS, 100 mM NaF, 2 mM NaPPi, 1% NP-40) supplemented with protease and phosphatase inhibitors. Lysates were centrifuged at 13,000 × g for 30 min at 4 °C and the resulting supernatants were subjected to immunoblot analysis with the following antibodies: mouse anti-Gli1 (L42B10, 1:500) and rabbit anti-PARP (9542, 1:1000) purchased from Cell Signaling (Beverly, MA, USA); rabbit anti-Cyclin D1-20 (sc-717, 1:500), rabbit anti-β-Catenin (sc-7199, 1:1000) and goat anti-Actin (sc-1616, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse anti-ERAP1 6H9 (1:1000) kindly provided by P. van Endert; goat anti-Gli2 (AF3635, 1:1000) was purchased from R&D Systems; mouse anti-Itch (611199, 1:1000) antibody was purchased from BD Bioscience (Heidelberg, Germany); rabbit anti-HDAC1 (H3284, 1:1000) was purchased from Sigma Aldrich (St. Louis, MO, USA).
3
Antibody Immunostaining Protocol for Organelles
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The primary antibodies used in this study were: rabbit anti-Rab7 (for IHC: ab 137029; Abcam, Cambridge, UK), chicken anti-Rab7 (for WB), rabbit anti-LC3 (for WB: PM036; MBL, Nagoya, Japan) (for IHC: #3868; Cell Signaling Technology), guinea pig anti-p62 (PM066; MBL), rat anti-N-terminal of lysosomal-associated membrane protein (LAMP)-1 (553792; BD Biosciences, San Jose, CA, USA) and rabbit anti-C-terminal of LAMP-1 (ab24170; Abcam), mouse anti-cathepsin B (AF965; R&D Systems, Minneapolis, MN, USA), rabbit anti-EEA1 (#2411; Cell Signaling Technology), mouse anti-CI-MPR (NB300–514; Novus, Littleton, CO, USA) and goat anti-actin (sc-1616; Santa Cruz Biotechnology, Dallas, TX, USA) immunoglobulins (IgGs). The secondary antibodies used in this study were: horseradish peroxidase-conjugated goat anti-guinea pig (Cosmo Bio, Tokyo, Japan), donkey anti-rabbit, anti-chicken and anti-mouse IgGs, and FITC- or Cy3-conjugated donkey anti-rabbit, anti-mouse and anti-rat IgGs (Jackson ImmunoResearch, West Grove, PA, USA).
4
Histological Analysis of Tissue Samples
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Samples were processed using standard histological protocols. Briefly, samples were fixed in methanol, dehydrated, and embedded in paraffin wax. Transverse sections (10 μm thick) were prepared and mounted on poly‐l ‐lysine coated slides. For Masson's trichrome stain a commercial kit was used (Bio‐Optica 04‐010802, Milan, Italy) on dewaxed and rehydrated sections. For immunofluorescence, dewaxed and rehydrated section tissues were subjected to antigen retrieval using 20 × 5 sec microwave pulses in sodium citrate (0.1 mol/L, pH 6), and blocked with 2% donkey serum. Primary antibodies used in this study were goat anti‐actin (sc‐1616, 1/250 dilution, Santa Cruz Biotechnologies, Dallas, TX, USA) and mouse anti‐β‐tubulin (TUJ‐1 clone, MMS‐435‐250, 1/1000 dilution, Covance, Burlington, NC, USA). Secondary antibodies used were donkey anti‐goat IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate (A‐11055, Life Technologies, Waltham, MA, USA), and donkey anti‐mouse IgG (H+L) secondary antibody, Alexa Fluor® 594 conjugate (Life Technologies, A‐21203).
5
Western Blot Analysis of Apoptosis Markers
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Western blot experiments were performed as previously described.33 (link) In brief, cells were lysed in RIPA buffer and 40 μg of total proteins were resolved in 4–12% gradient gels by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and incubated with rabbit anti-poly ADP-ribose polymerase (11835238001, Roche Diagnostics GmbH, Mannheim, DE, USA), active caspase 3 (559565, BD Biosciences, St Jose, CA, USA) and goat anti-actin (Sc1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. Protein lysates from tumors were probed with poly ADP-ribose polymerase cleavage antibody (AB3565, Millipore, Billerica, MA, USA) and HSP72/73 antibody (HSP01, Millipore). The membranes were subsequently probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and antigens on the membrane were revealed by enhanced chemiluminescence (RPN2209, GE Healthcare, Little Chalfont, UK).
6
Quantitative Analysis of MYPT1 and RhoA Signaling
7
Western Blotting of LIN28B and Actin
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The following antibodies were used for Western blotting: rabbit anti-LIN28B (4196; Cell Signaling Technology) and goat anti-actin (sc-1616; Santa Cruz Biotechnology, Inc.).
8
Quantitative Analysis of MYPT1 and RhoA Signaling
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NIH/3T3 cells were rinsed once with PBS and lysed in M-PER lysis buffer (Thermo Fisher) supplemented with protease and phosphatase inhibitor cocktail (Sigma). Protein concentration of the lysates cleared of insoluble cell debris were determined using 660 nm Protein Assay reagent (Thermo Fisher). A total of 15 μg of proteins in LDS electrophoresis loading buffer (Life Technologies) was denatured for 10 min at 70 °C and separated on 4–12% SDS-PAGE gel (Life Technologies). Proteins were transferred onto 0.2-μm nitrocellulose membrane (Pall) and processed for Western blotting. Primary antibodies were used at the following dilutions: goat anti-actin (sc-1616, Santa-Cruz Biotechnology, 1:4,000), mouse anti-MYPT1 (612165, Becton-Dickinson, 1:4,000), rabbit anti-MYPT1(pT696) (ABS45, Millipore, 1:500), rabbit anti-RhoA (67B9, Cell Signaling, 1:4,000), rabbit anti-MLC2 (8505, Cell Signaling, 1:4,000), and mouse anti-MLC2(pT19) (3674, Cell Signaling, 1:500). Appropriate secondary IRDye-conjugated antibodies (LI-COR) were used at 1:10,000. Proteins were detected using Odyssey imager (LI-COR). The investigator carrying out the Western blot experiments was not blinded to the identity of the samples.
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