A portion of the CM prepared for MS was saved for Western blotting analysis. The protein concentration of each sample was determined using the bicinchoninic acid (BCA) assay (Thermo Scientific) with bovine serum albumin (BSA) as a standard. 25 μg protein was loaded onto each lane of a denaturing 4–12% gradient gel and fractionated. Proteins were transferred to a nitrocellulose membrane and the blots were probed with a CTSD- (sc10725, Santa Cruz Biotechnologies), ECM 1- (ab126629, Abcam), PRDX1- (ab109498, Abcam), or SFN- (ab14123, Abcam) specific antibody. The SFN sample was concentrated up to 6-fold using Amicon Ultra-0.5 Centrifugal Filter 3K Devices (Millipore). Western blots were imaged and quantitated with a Licor Odyssey Infrared Imaging System.
Ab109498
Manufactured by Abcam
Sourced in United Kingdom
Ab109498 is a polyclonal antibody that specifically recognizes the target protein. It is intended for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab16830, by Abcam (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Ab14123, by Abcam (1 mentions)
Ab126629, by Abcam (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Sc 10725, by Santa Cruz Biotechnology (1 mentions)
Ab184247, by Abcam (1 mentions)
Ab115777, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab65556, by Abcam (1 mentions)
Ab3191, by Abcam (1 mentions)
Ab191217, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab202068, by Abcam (1 mentions)
Ab25758, by Abcam (1 mentions)
4 protocols using ab109498
1
Western Blot Analysis of CTSD, ECM1, PRDX1, SFN
2
Protein Expression Analysis in HepG2 Cells
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Lysis buffer (200 μl/well) was used to lyse HepG2 cells. Lysis buffer was composed of
Triton X-100 (1%), Tris (50 mM, pH=7.6) and NaCl (150 mM), with inhibitors of phosphatases
and proteases. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 7.5%)
was used to separate 40 μg of the total extracted protein. Then Western blotting was done
as demonstrated by Moeschel et al. (10 (link)). Following the application of antibodies for
western blotting anti-PRDX1 (ab109498), anti-beta actin (ab115777), anti
caspase-3 (ab13847 and ab32042), anti-cleave caspase-9 (ab202068 and ab25758), anti-PARP-1
(ab191217), anti-Bim (ab7888), antiFis1 (ab189846), anti-APaf-1 (ab254248), anticytochrome
c (ab133504), anti-Bcl-2 (ab182858), anti-Bax (ab3191), anti-DRP1 (ab184247) and antiDyn2
(ab65556; all purchased from AbCam, UK). Nitrocellulose was blocked using skimmed milk
(5%) or BSA (2%, both from Merck, Germany) for two hours. Subsequently, membranes were
incubated with primary antibodies at 4°C overnight. Before incubation with secondary
antibody, washing was performed (four times for 10 minutes), followed by appropriate
conjugated secondary incubation for one hour. For visualizing expression level of
proteins, enhanced chemiluminescence was performed.
Triton X-100 (1%), Tris (50 mM, pH=7.6) and NaCl (150 mM), with inhibitors of phosphatases
and proteases. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 7.5%)
was used to separate 40 μg of the total extracted protein. Then Western blotting was done
as demonstrated by Moeschel et al. (10 (link)). Following the application of antibodies for
western blotting anti-PRDX1 (ab109498), anti-beta actin (ab115777), anti
caspase-3 (ab13847 and ab32042), anti-cleave caspase-9 (ab202068 and ab25758), anti-PARP-1
(ab191217), anti-Bim (ab7888), antiFis1 (ab189846), anti-APaf-1 (ab254248), anticytochrome
c (ab133504), anti-Bcl-2 (ab182858), anti-Bax (ab3191), anti-DRP1 (ab184247) and antiDyn2
(ab65556; all purchased from AbCam, UK). Nitrocellulose was blocked using skimmed milk
(5%) or BSA (2%, both from Merck, Germany) for two hours. Subsequently, membranes were
incubated with primary antibodies at 4°C overnight. Before incubation with secondary
antibody, washing was performed (four times for 10 minutes), followed by appropriate
conjugated secondary incubation for one hour. For visualizing expression level of
proteins, enhanced chemiluminescence was performed.
3
Peroxiredoxin Protein Analysis Protocol
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Cells (approx.100,000 cells/dish) were plated to a 6 cm dish and incubated for 48 hours. They were washed with PBS, scraped off the plate, centrifuged and the cell pellet was lysed using a Lysis Buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, and 0/1% SDS in water + protease inhibitor cocktail [Sigma] + phosphatase inhibitor cocktail [Sigma-Aldrich] + okadaic acid + sodium fluoride). The cells were spun down and the supernatant was used to measure protein concentration by using a Bradford Assay (BioRad). Equal protein concentrations were loaded onto a NuPAGE 4–12% Bis-Tris gels (Invitrogen). Protein was transferred to a nitrocellulose membrane and incubated in blocking solution (PBS, 5% BSA, 0.1% Tween 20) for 1 hour at room temperature. The membrane was incubated with primary antibodies at 4°C overnight, rinsed three times with PBST and incubated with secondary antibodies for 1 hour at room temperature. The blot was imaged on the LICOR Odyssey. Primary Antibodies used: Anti-Peroxiredoxin-SO3 antibody (ab16830) (1:1000), Recombinant Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) (1:1000) from Abcam and actin (Sigma) was used for primary staining. Secondary antibodies used: 680LT secondary (LICOR-IRDye) and 800CW secondary (LICOR-IRDye), both at 1:10000 concentration.
4
Quantifying Cellular Oxidative Stress
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Cells (approx.100,000 cells/dish) were plated to a 6 cm dish and incubated for 48 h. They were washed with PBS, scraped off the plate, centrifuged and the cell pellet was lysed using a Lysis Buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, and 0.1% SDS in water + protease inhibitor cocktail [Sigma] + phosphatase inhibitor cocktail [Sigma-Aldrich] + okadaic acid + sodium fluoride). The cells were spun down and the supernatant was used to measure protein concentration by using a Bradford Assay (BioRad). Equal protein concentrations were loaded onto a NuPAGE 4-12% Bis-Tris gels (Invitrogen). Protein was transferred to a nitrocellulose membrane and incubated in blocking solution (PBS, 5% BSA, 0.1% Tween 20) for 1 h at room temperature. The membrane was incubated with primary antibodies at 4 oC overnight, rinsed three times with PBST and incubated with secondary antibodies for 1 h at room temperature. The blot was imaged on the LI-COR Odyssey. Primary Antibodies used: Anti-Peroxiredoxin-SO3 antibody (ab16830) (1:1000), Recombinant Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) (1:1000) from Abcam and actin Cat#A2228 clone AC-74 (Sigma) was used for primary staining. Secondary antibodies used: 680LT secondary LICOR-IR Dye Cat# 925-68020, and 800CW secondary LICOR-IR Dye Cat# 925-3221, both at 1:10000 concentration.
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