Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting were performed as described previously [33 (link)]. Chemiluminescent signals were developed using ECL and visualized with GeneTools (Syngene, Bangalore, India). Primary antibodies against PARP1 (C2-10), β-actin, SQSTM1 (A-6), phospho-p70 S6 kinase (Ser434), p70 S6 kinase (Santa Cruz Biotechnology, Dallas, Texas, USA), caspase-3, AMPK, phospho-AMPK (Thr172), and LC3 (Novus Biologicals, Centennial, CO, USA) were used. Secondary antibodies were HRP-conjugated swine anti-rabbit and HRP-conjugated rabbit anti-mouse (Agilent, Santa Clara, CA, USA).
Hrp conjugated rabbit anti mouse
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The HRP-conjugated rabbit anti-mouse is a secondary antibody used in various immunoassay techniques. It is composed of a rabbit-derived antibody that specifically binds to mouse primary antibodies, conjugated with the enzyme horseradish peroxidase (HRP). This conjugation allows for the detection and quantification of target antigens in samples through colorimetric or chemiluminescent readouts.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated swine anti rabbit, by Agilent Technologies (5 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Caspase 3, by Novus Biologicals (1 mentions)
Pd l1 e1l3n xp, by Cell Signaling Technology (1 mentions)
P53 do 1, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Digital imaging system, by Nikon (1 mentions)
Vimentin, by Agilent Technologies (1 mentions)
E cadherin, by Agilent Technologies (1 mentions)
Snai2, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Abcam (1 mentions)
α tubulin, by Merck Group (1 mentions)
Alexa fluor 532 goat anti rabbit igg, by Abcam (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
Hecd 1, by Abcam (1 mentions)
P smad2, by Cell Signaling Technology (1 mentions)
Smad4, by Cell Signaling Technology (1 mentions)
Hrp conjugated mouse anti β actin, by Merck Group (1 mentions)
G box chemi xx6, by Syngene (1 mentions)
11 protocols using hrp conjugated rabbit anti mouse
1
Protein Expression Analysis via SDS-PAGE
2
Protein Expression Analysis by Immunoblotting
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Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting were performed as described previously [34 (link)]. Primary antibodies against PD-L1 (E1L3N) XP (Cell Signaling Technology), CD276 (B7-H3) (R&D Systems Inc.), MDM2 (4B2) [35 (link)], p53 (DO-1) [36 (link)], β-actin and GAPDH (both Santa Cruz Biotechnology) were used. Secondary antibodies were HRP-conjugated swine anti-rabbit, HRP-conjugated rabbit anti-mouse (both Agilent) and HRP-conjugated donkey anti-goat (Abcam).
3
Western Blot Analysis of Protein Expression
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Cells were washed twice with cold phosphate-buffered saline (PBS) and then scraped into NET lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris pH 8.0, 50 mM NaF, 5 mM EDTA pH 8.0) supplemented with protease and phosphatase inhibitor cocktails according to the manufacturer’s instructions (Sigma-Aldrich-St. Louis, MO, USA). Following SDS-PAGE, samples were transferred onto nitrocellulose membranes and incubated overnight at 4 °C with primary antibodies. The following day, membranes were washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000) for 1 h at room temperature (RT). Chemiluminescent signals were developed using ECL solution and visualized with GeneTools (Syngene). Actin was used as a loading control and as a reference for normalization. Antibodies: AGR2 rabbit polyclonal serum (K-31, in-house); actin monoclonal antibody (ACTN05 C4), COL4A1 Polyclonal Antibody (PA585634), VAV3 Polyclonal Antibody (PA5113523) (all ThermoFisher Scientific, Waltham, MA, USA), PTGS2 antibody (HPA001335, Sigma-Aldrich, St. Louis, MO, USA), HRP-conjugated swine anti-rabbit, and HRP-conjugated rabbit anti-mouse (both Dako, Agilent, Santa Clara, CA, USA). Statistical significance was calculated using One-Way ANOVA test with Post Hoc Tukey HSD.
4
In vivo Expression of Target Antigen
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Example 4
For the in vivo transduction analysis, three mice from Bac-VP1, wild type baculovirus and PBS immunized group were euthanized on day 6, and muscle tissues were collected in 10% (wt/vol) buffered formalin, embedded in paraffin, and sectioned. The sections were then deparaffinized using Histo-choice (Amersco) and rehydrated in sequentially graduated ethanol baths. Slides were blocked in 0.3% nonfat milk in PBS for 30 min, followed by incubation with anti-VP1 monoclonal antibody for 1 h at 37° C. Slides were then washed 3 times in PBS and incubated with HRP-conjugated rabbit anti-mouse (Dako Cytomation, Denmark) at a dilution of 1:50 for 30 min. After washing, the slides were incubated with hematoxylin for 2-5 min and the slides were washed thrice with sterile water and the sections were mounted using mounting medium. The slides were observed under a microscope (Olympus, UK) and the images were captured by digital imaging system (Nikon, USA) (
As can be seen from
5
Verification of Antibody Specificity in Testicular Samples
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Western blotting (WB) was carried out as previously (Sonne et al, 2006 (link); Jørgensen et al, 2013b (link)) to verify antibody specificity. Samples included normal adult testis (NT), testis with CIS and the absence of complete spermatogenesis (CIS), and seminoma samples (SEM) (Supplementary Figure 1 ). Primary antibodies and dilutions are listed in Table 1 and 10 μg protein was loaded in each lane. Detection of β-actin was used as a loading control based on a stable expression in normal testis tissue, tissue containing CIS and seminoma tissue (Jørgensen et al, 2013b (link)). Secondary antibodies were HRP-conjugated rabbit anti-mouse (Dako, Glostrup, Denmark, P0260) and HRP-conjugated swine anti-rabbit (Dako, P0217), both diluted 1 : 1000. All antibodies appeared specific, with only one band detected at the expected size, except for the anti-AP2γ, which detected two bands in CIS and seminoma samples (48 and 40 kDa), and only one faint band (48 kDa) in the NT sample that was expected to be negative. The signals corresponding to PLAP, AP2γ and KIT were much more intense in seminoma samples compared with normal testis and samples with CIS (Supplementary Figure 1 ).
6
Comprehensive Protein Expression Analysis in EMT
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Akt2 (5B5), p-AKT (S473; 736E11), p44/42 MAPK (Erk1/2; 137F5), p-p44/42 MAPK (T202/Y204, D13.14.4E), p-Smad2 (S465/467; 138D4), Smad4, SNAI2, ZEB1 (all Cell Signaling Technology); AGR2 (K-31, in-house); AGR2 (1C3, Abnova); vimentin (V9, Dako); E-cadherin (NCH38, Dako; HECD1, Abcam; Cell Signaling); N-cadherin (3B9, Invitrogen); β-actin (C4, Santa Cruz Biotech); Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 532 goat anti-rabbit IgG (both Abcam); α-tubulin (AA13, Sigma); HRP-conjugated swine anti-rabbit and HRP-conjugated rabbit anti-mouse (both Dako).
7
Western Blot Analysis of LRRC8A
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For western blots, cells were lysed directly in laemmli buffer (62.5 mM Tris–HCl, 2% SDS, 10% glycerol, 5% β‐mercaptoethanol) and heated to 95°C for 10 min. Lysates were then separated by tris‐glycine SDS‐PAGE before transfer to PVDF membranes. Membranes were blocked in 5% nonfat milk in phosphate‐buffered saline (PBS) with 0.05% Tween‐20 (PBS‐T) for 1 h at room temperature before incubating overnight with mouse anti‐LRRC8A (8H9, Santa Cruz, 1:100) in block buffer. Membranes were washed three times in PBS‐T before incubating with HRP‐conjugated rabbit anti‐mouse (Dako) in block buffer for 1 h at room temperature. After washing three further times, membranes were imaged on a G:Box Chemi XX6 (Syngene) using Amersham ECL Prime chemiluminescence reagent (GE Healthcare).
For loading controls, membranes were washed and incubated with HRP‐conjugated mouse anti‐β‐actin (Sigma) in block buffer for 1 h at room temperature, followed by washing and imaging as above.
For loading controls, membranes were washed and incubated with HRP‐conjugated mouse anti‐β‐actin (Sigma) in block buffer for 1 h at room temperature, followed by washing and imaging as above.
8
ELISA Binding Assay of RSV F Protein
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96-well Nunc maxisorp plates were coated with different F protein preparations (100 ng/well) o/n at 4°C. After blocking (phosphate buffered saline [PBS] with 0.1% Tween-20 v/v and 3% bovine serum albumin w/v) and washing (PBS with 0.05% Tween-20 v/v), the plates were incubated with 2-fold serial dilutions of StrepMab-classic (IBA, start dilution 1:200), Palivizumab (start dilution 1:1000), AM22 (start dilution 1:200), D25 (start dilution 1:200), α6HB [17 (link),22 (link)](start dilution 1:100) and 131-2a (Millipore, start dilution 1:2000) using 1mg/ml antibody stocks. After extensive washing, the plates (except for the HRP-conjugated polyclonal goat anti-RSV incubated plates) were incubated with 1:500 dilution of either HRP-conjugated rabbit anti-mouse (Dako), goat anti-human (ITK Southern Biotech) or goat anti-rabbit (Biorad) IgG antibodies for one hour at room temperature. Detection of HRP reactivity was performed using tetramethylbenzidine substrate (BioFX) and an ELISA plate reader (EL-808 from Biotek). All experiments were performed at least three times with independently generated F protein preparations. The mean OD450nm values are depicted in the graphs. One way ANOVA (Graphpad software) was performed using the OD450nm values corresponding to a single dilution of antibody within the linear part of the ELISA curves (S2 , S3 and S6 Figs).
9
Aβ1-42 oligomer detection by dot blot
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2 μl of the respective Aβ1-42 solution was applied onto nitro-cellulose membranes. The membranes were incubated with either oligomer-specific (A11, 1:200, Invitrogen) or Aβ1-42 –specific (4G8, 1:200, Covance) antibodies o/n. Membranes were then washed and blocked with 5 % skim milk/PBS for 1 h at RT. Secondary antibodies for dot blot were HRP-conjugated rabbit-anti-mouse (1:1000, DAKO) and HRP-conjugated goat-anti-rabbit (1:1000, DAKO). Spots were visualized by ECL (Amersham) and subsequent exposure to X-ray films.
10
Western Blot Analysis of Cellular Proteins
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Cell extracts were mixed with Laemmli buffer to a final concentration of 5 million cell-equivalents/15 μl. Either one million or five million cell-equivalents (depending on the experiment) were loaded per lane on an SDS-polyacrylamide gel. The gels were resolved by 80-120V and transferred (wet transfer, 100V for 1h) onto PVDF Immobilon- FL transfer membranes (0.45 μm, MILLIPORE), followed by blocking (5% milk in PBS + 0.1% Tween-20 (PBST)) for 1 h at room temperature. Primary antibodies were incubated for either 1 h at room temperature or overnight at 4°C. Secondary antibodies were applied for 1 h at room temperature. All used antibodies were diluted in blocking solution. The following antibodies were used: mouse anti-TAC102 (1:1000, [24 (link)]), rabbit anti-HA (1:1000, Sigma-Aldrich), rabbit anti-ATOM40 (1:10000, [61 (link)]), rabbit anti-mouse HRP-conjugated (1:10000, Dako) and swine anti-rabbit HRP-conjugate (1:10000, Dako), rabbit peroxidase anti-peroxidase soluble complex (PAP) (1:2000, for detection of the PTP tag). After antibody incubation, the membranes were washed three times with PBST. A final wash with PBS was performed prior to detection of the protein with the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). The Amersham Imager 600 (GE Healthcare) was used to visualize the protein bands on the blot.
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