The largest database of trusted experimental protocols

Mouse anti actin

Manufactured by Thermo Fisher Scientific
Sourced in United States

Mouse anti-actin is a primary antibody that specifically binds to the actin protein, which is a key structural component found in eukaryotic cells. This antibody can be used for the detection and analysis of actin in various research and diagnostic applications.

Automatically generated - may contain errors

18 protocols using mouse anti actin

1

Western Blot Analysis of HA-Tagged Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Late trophozoite parasites cultured with or without aTc were isolated using a 65% Percoll gradient. Recovered parasites were resuspended in Laemmli Sample Buffer (Bio-rad) and β-mercaptoethanol (Sigma-Aldrich) and loaded on a 10% Mini-PROTEAN® TGX™ Precast Protein Gel. Samples were transferred to 0.2 μm polyvinylidene difluoride (PVDF) membrane using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked in 5% skim milk in 0.1% PBS-Tween (PBS-T) for 1 h at room temperature and incubated overnight with rat anti-HA tag (Roche) and mouse anti-Actin (Invitrogen) at 1:3000 in 2% Bovine Serum Albumin (BSA). The blot was then washed 3 times in PBS-T and probed with goat anti-rat HRP (Biolegend) and goat anti-mouse HRP (Biolegend) at 1:10,000 in 2% BSA PBS-T for 1 h at room temperature. The blot was imaged on ChemiDoc MP (Bio-Rad) in Clarity Max Western ECL Substrate (Bio-Rad). Western blot analysis was done using Image Lab v6.0 (Bio-Rad).
+ Open protocol
+ Expand
2

Protein Expression Profiling Using Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols
To assess expression of endogenous or transduced proteins, cell lysates containing 30–40 μg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: mouse anti-TMPRSS2 (Santa Cruz, #515727, 1:1000), mouse anti-Cathepsin-L (Santa Cruz, #32320, 1:1000), goat anti-ACE-2 (R&D systems, #AF933, 1;1000), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-MAVS (Thermo Fisher, #PA5–17256, 1:1000), mouse anti-RIG-I (AdipoGen, #20B-0009, 1:1000), rabbit anti-MDA-5 (Proteintech, #21775–1-AP, 1:1000), rabbit anti-UNC93B1 (Invitrogen, #PA5–83437, 1:1000), rabbit anti-IRF1 (Cell Signaling, #8478S, 1:1000). Specific staining was visualized with secondary antibodies, goat anti-mouse-IgG-DyLight 680 (Thermo Scientific, #35518, 1:20000), goat anti-rabbit-IgG-DyLight 800 (Thermo Scientific, #SA5–35571, 1:20000), or a donkey anti-goat-IgG-IR-Dye 800 (Licor, #926–32214, 1:20000). As loading controls, actin or tubulin expression was probed using a rabbit anti-actin (Sigma-Aldrich, A2066, 1:5000), mouse anti-actin (Invitrogen, #AM4302, 1:5000), or rabbit anti-tubulin (Cell Signaling, #3873, 1;5000). Membranes were scanned with an Odessy scanner (Li-Cor).
+ Open protocol
+ Expand
3

Quantification of Polyglutamine Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
For polyQ protein quantification, treated young adult animals were washed with M9 1X buffer before lysing the samples with RIPA buffer (Invitrogen, Carlsbad, CA, USA) and proteinase inhibitors cocktail (Complete, Roche, Basel, Switzerland). Samples were boiled at 100 °C for 10 min containing 4X SDS sample loading buffer. Protein extracts were separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene di-fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA) by semi-dry blotting (Trans-Blot Turbo, Bio-Rad). Blocking was done with 3% milk, according to the specification of the following primary antibodies: mouse anti-polyQ (1:1000, Sigma Ref. #P1874) and mouse anti-actin (1:500, Invitrogen ref. #MA5–11869) to normalize values. Primary antibodies were incubated overnight at 4 °C degrees with shaking. We used the secondary antibody anti-mouse conjugated to HRP (1:10000, Santa Cruz ref. #SC-2005, Santa Cruz Biotechnology, Dallas, TX, USA) to develop immunoblots. Images were obtained using Amersham Imager 600 and enhanced chemiluminescent (ECL) detection (GE Healthcare, Chicago, IL, USA). Quantification values were obtained using the Image J software.
+ Open protocol
+ Expand
4

Secretion Assay for Ustilago maydis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Secretion assay was performed as described by Djamei et al. (2011). In brief, after filament induction, the cell‐free supernatant was precipitated with trichloroacetic acid (TCA)/Sodium deoxycholate (DOC) and a Western blot was performed on the precipitate. Ustilago maydis strain AB33 PotefapB73‐HA was generated by insertion of plasmid p123‐Potef‐ApB73‐HA into the ip locus of AB33 (Brachmann et al., 2001). AB33 cell extracts and supernatants were used to ensure specificity of the antibodies. Actin detection served as a lysis control and bands could only be observed for whole cell extracts. For Western blot analysis, mouse anti‐haemagglutinin (anti‐HA) (Sigma‐Aldrich, St. Louis, MO, USA) and mouse anti‐actin (Invitrogen, Waltham, MA, USA) antibodies were used. Assays were carried out at least three times.
+ Open protocol
+ Expand
5

Quantification of Polyglutamine Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
For polyQ protein quantification, treated young adult animals were washed with M9 1X buffer before lysing the samples with RIPA buffer (Invitrogen, Carlsbad, CA, USA) and proteinase inhibitors cocktail (Complete, Roche, Basel, Switzerland). Samples were boiled at 100 °C for 10 min containing 4X SDS sample loading buffer. Protein extracts were separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene di-fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA) by semi-dry blotting (Trans-Blot Turbo, Bio-Rad). Blocking was done with 3% milk, according to the specification of the following primary antibodies: mouse anti-polyQ (1:1000, Sigma Ref. #P1874) and mouse anti-actin (1:500, Invitrogen ref. #MA5–11869) to normalize values. Primary antibodies were incubated overnight at 4 °C degrees with shaking. We used the secondary antibody anti-mouse conjugated to HRP (1:10000, Santa Cruz ref. #SC-2005, Santa Cruz Biotechnology, Dallas, TX, USA) to develop immunoblots. Images were obtained using Amersham Imager 600 and enhanced chemiluminescent (ECL) detection (GE Healthcare, Chicago, IL, USA). Quantification values were obtained using the Image J software.
+ Open protocol
+ Expand
6

Western Blot Analysis of BPNT2 Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols
Protein was collected from the cells lysed in RIPA buffer with the protease inhibitor (Roche). Protein extracts were passed through a 25g needle to break up DNA and subsequently quantified using the BCA assay. Ten microgram of total protein was loaded per lane onto a 12% SDS-PAGE gel (Bio-Rad), which was run at 100 V for 2 h. The proteins were transferred to a 0.2-μm PVDF membrane (Bio-Rad) using Trans-Blot Turbo (Bio-Rad) at 1.3 A for 7 min. Blots were incubated in primary antibodies (sheep anti-BPNT2, 1:1000, Invitrogen, #PA5-47893; mouse anti-Actin, 1:1000, Invitrogen, #MA1-744) overnight at 4 °C, washed 3 times in 0.1% TBS-Tween, then in secondary antibodies (AlexaFluor680 anti-sheep 1:20,000; AlexaFluor800 anti-mouse 1:20,000) for 2 h at room temperature, and washed 3 times in 0.1% TBS-T. The blots were imaged on LI-COR Odyssey.
+ Open protocol
+ Expand
7

Western Blot Analysis of Antiviral Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
To assess expression of endogenous or transduced proteins, cell lysates containing 30–40 μg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: mouse anti-TMPRSS2 (Santa Cruz, #515727, 1:1000), mouse anti-Cathepsin-L (Santa Cruz, #32320, 1:1000), goat anti-ACE2 (R&D systems, #AF933, 1;1000), rabbit anti-STING (Cell Signaling, #13647, 1:1000), rabbit anti-MAVS (Thermo Fisher, #PA5-17256, 1:1000), mouse anti-RIG-I (AdipoGen, #20B-0009, 1:1000), rabbit anti-MDA-5 (Proteintech, #21775-1-AP, 1:1000), rabbit anti-UNC93B1 (Invitrogen, #PA5-83437, 1:1000), rabbit anti-IRF1 (Cell Signaling, #8478S, 1:1000). Specific staining was visualized with secondary antibodies, goat anti-mouse-IgG-DyLight 680 (Thermo Scientific, #35518, 1:20000), goat anti-rabbit-IgG-DyLight 800 (Thermo Scientific, #SA5-35571, 1:20000), or a donkey anti-goat-IgG-IR-Dye 800 (Licor, #926–32214, 1:20000). As loading controls, actin or tubulin expression was probed using a rabbit anti-actin (Sigma-Aldrich, A2066, 1:5000), mouse anti-actin (Invitrogen, #AM4302, 1:5000), or rabbit anti-tubulin (Cell Signaling, #3873, 1:5000). Membranes were scanned with an Odessy scanner (Li-Cor).
+ Open protocol
+ Expand
8

Tubulin Isoforms Characterization by Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols
Protein extracts were prepared from heads or dissected brains. Cell homogenization was done in RIPA buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0, supplemented with protease inhibitor cocktail from Roche Life Science). After 2 hrs of lysis at room temperature, Laemmli buffer was added before to boil the samples during 5 min.
Samples were analyzed by SDS-PAGE. To separate alpha and beta-tubulins, we used a protocol described by Banerjee and collaborators63 (link). Separated proteins were electrophoretically transferred onto nitrocellulose membrane (Hybond C-Extra, Amersham Biosciences) prior to blotting. Primary and secondary antibodies were incubated in 5% milk in PTX (PBS, 0.1% TritonX100) and washes were done with PTX. Immunodetection was done with Clarity Western ECL kit (BIO-RAD). Chemiluminescence detection was acquired with ChemiDoc Touch Imaging System (BIO-RAD). The following primary antibodies were used: mouse GT335 (1/200, AdipoGen), mouse 1D5 (1/500, Synaptic System), mouse anti-acetylated-tubulin (1/2000, Sigma), mouse DM1A (1/4000, anti-alpha tubulin from Sigma), mouse E7 (1/1000, anti-beta tubulin from DSHB), mouse anti-actin (1/2000, ThermoScientific). Secondary antibody used was HRP-linked goat anti-mouse (1/10000, Jackson ImmunoResearch).
+ Open protocol
+ Expand
9

Cell Vulnerability Assays for Huntington's Disease

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell vulnerability assays were performed as it follows. Low-passage normal (STHdh7Q) and mutant (STHdh109Q) Htt mouse striatal cells20 (link) were cultured in 24-well plates and cell vulnerability assays performed as previously described using serum deprivation for cell death induction97 (link). Serum deprivation was performed by replacing the medium with 1% FBS medium and with drug or vehicle. Cell vulnerability was scored 24 hours later upon DAPI staining and by counting pyknotic versus normal nuclei in DAPI-positive cells. This involved three independent experiments with 100 scored cells each, and assays were performed blindly. For Western blotting, proteins were extracted as previously described97 (link), separated by SDS-PAGE, and analyzed by Western blotting using the following primary antibodies: mouse anti-HTT (4C8, Chemicon, 1:5,000) and mouse anti-actin (ThermoFisher, 1:5,000). The secondary antibody used was goat–anti-mouse IgG HRP-conjugated (Biorad, 1:10,000). Proteins were detected by using ECL + (ECL for actin) and evaluated by densitometry. At least three independent assays were performed. Signals were quantified using ImageJ.
+ Open protocol
+ Expand
10

Immunoblotting Protein Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
For immunoblotting, cells were lysed in Cell Lysis Buffer (Cell Signaling Technology), and 10 μg of protein was subjected to SDS–polyacrylamide gel electrophoresis [using 4 to 12% (w/v) gradient gels (Life Technologies, NuPAGE Novex, Bis-Tris)], transferred to nitrocellulose membranes, and assayed by immunoblotting. The primary antibodies were mouse anti-actin (Thermo Fisher Scientific, β-actin loading control, 1:5000) and mouse anti-myosin heavy chain (R&D Systems, 1:2000). The secondary antibody was horseradish peroxidase (HRP)–conjugated anti-mouse IgG antibody (Cell Signaling Technology, Danvers, MA, USA). An HRP substrate was used for chemiluminescent analysis (Bio-Rad, Hercules, CA, USA).
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!