Samples treated with 2-mercaptoethanol were separated on 10% SDS-PAGE gels and then transferred onto nitrocellulose membranes (Millipore) by semi-dry transfer method. Membranes were blocked with 7% skimmed milk for an hour and incubated with primary antibodies overnight at 4 degree. The primary antibodies used in this study include HRP conjugated goat anti-human Fc (1:5000, Southern Biotech), mouse anti-HA mAb (HA.11, 1:1000, Covance), mouse anti-myc mAb (9B11, 1:1000, Cell signaling), mouse anti-FLAG M2 mAb (1:1000, Sigma), rabbit anti-γ-Tubulin (AK-15) antibody (1:1000, Sigma). After three washes with TBS-1% Tween-20, membranes were incubated with secondary antibodies including HRP conjugated goat anti-mouse Ig (1:10000, Dako), HRP conjugated goat anti-rabbit Ig (1:10000, Dako). After three washes, blots were developed using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) for 5 minutes.
Hrp conjugated goat anti mouse ig
Manufactured by Agilent Technologies
Sourced in Germany
HRP-conjugated goat anti-mouse Ig is a laboratory reagent used for the detection of mouse immunoglobulins in various immunoassay techniques. It consists of goat-derived antibodies specific to mouse immunoglobulins, conjugated with horseradish peroxidase (HRP) enzyme. The HRP label enables the visualization and quantification of target mouse immunoglobulins in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit ig, by Agilent Technologies (3 mentions)
Hrp conjugated goat anti human c3, by MP Biomedicals (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti flag m2 mouse mab, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha mab ha 11, by Fortrea (1 mentions)
Goat anti mouse ig, by Agilent Technologies (1 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine dihydrochloride, by Merck Group (1 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Hrp conjugated anti human igg, by Merck Group (1 mentions)
Purified human fh, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by AppliChem (1 mentions)
5 protocols using hrp conjugated goat anti mouse ig
1
Western Blotting of Protein Samples
2
Proteolytic activity of Sapp1p and Sapp2p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteolytic activity of purified Sapp1p and Sapp2p (1 μg each) was assayed by incubating them with purified human complement proteins C3b, C4b, and factor H (FH) (Merck) or with recombinant factor H-like protein 1 (FHL-1) (expressed and purified as described previously) (66 (link)) or FHR-1 or FHR-5 (Novoprotein) for 3 h or 15 h at 37°C. Aliquots were taken at the indicated time points, separated by SDS-PAGE, and analyzed by Western blotting. C3b was identified by the use of polyclonal goat anti-human C3 (Calbiochem, Quidel) in combination with a horseradish peroxidase (HRP)-conjugated goat antibody (DAKOCytomation). C4b was detected with a monoclonal anti-C4c antibody (Quidel) and with HRP-conjugated goat anti-mouse Ig (Dako). To detect cleavage of FH, FHL-1, FHR-1, and FHR-5, polyclonal goat anti-FH (Calbiochem, Merck), mouse monoclonal anti-FH (A254; from Quidel), and polyclonal goat anti-FHR-5 (R&D System) and the corresponding HRP-conjugated secondary antibodies rabbit anti-goat Ig and goat anti-mouse Ig (Dako) were used. In addition, cleavage of C3b and C4b by the natural, complement-specific protease factor I in the presence of the cofactors factor H and C4BP (Hyphen Biomed) was assayed to compare with the cleavage patterns generated by the Sapp proteases.
3
Quantifying c-Abl SH2 Domain Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We measured the capacity of the c-Abl–SH2 domain to bind to synthetic peptides using ELISA. Briefly, synthetic peptides (50 μg/ml) (biotin-GGA-STYPKSPDSRQPEPLY [*p] ENVNVVSGNEVYSLV and biotin-GGA-STYPKSPDSRQPEPLYENVNVVSGNEVYSLV) were diluted fourfold in PBS buffer to create a concentration gradient and coated onto Nunc MaxiSorp ELISA plates overnight at 4°C. The plates were further blocked with 0.3% (w/v) gelatin in PBS buffer for 2 hours at 37°C, followed by the addition of GST (10 μg/ml) or GST-SH2 fusion proteins for 1 hour at 37°C. The plates were then washed with PBST buffer (PBS with 0.05% Tween 20) and incubated with mouse anti-GST antibody (clone N100/13, NeuroMab) for 1 hour at room temperature. After washing, plates were incubated with horseradish peroxidase (HRP)–conjugated goat anti-mouse Ig (00090941, Dako) as the second antibody for 45 min at room temperature. After washing, the substrate solution, comprising 0.325% o-phenylenediamine dihydrochloride (Sigma-Aldrich) and 0.085% H2O2 in 0.3 M tris-citrate buffer (pH 6.0), was added. After incubation at room temperature for 10 min in the dark, the reaction was stopped with 2.5 M H2SO4, and the plates were measured using an ELISA plate reader (Bio-Rad) for light absorbance at 490 nm.
4
Purification and Expression of Complement Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified human FH, FB, C3b, factor D, C1q, goat anti-human C1q antibody (Ab) and goat anti-human FH antibody (Ab) were purchased from Merck (Budapest, Hungary). Human serum albumin (HSA), bovine serum albumin (BSA), alpha1-antitrypsin, HRP-conjugated anti-human IgG, HRP-conjugated anti-human IgA, HRP-conjugated anti-human IgM, and monoclonal antibodies (mAbs) specific for IgG1, IgG2, IgG3, IgG4, Ig kappa and Ig lambda were purchased from Sigma-Aldrich (Budapest, Hungary). HRP-conjugated goat anti-mouse Ig and HRP-conjugated rabbit anti-goat Ig were purchased from DakoCytomation (Hamburg, Germany). HRP-conjugated goat anti-human C3 was from MP Biomedicals (Solon, OH). The anti-FH mAb A254 was purchased from Quidel (Biomedica, Budapest, Hungary), and the mAb C18 (40 (link)) was from Alexis Biochemicals (Lörrach, Germany). The anti-FH mAb IXF9 was described earlier (41 (link)).
Codon-optimized sequences of FHR-1, FHR-4B, FH SCRs 1-4, FH SCRs 8-14, FH SCRs 15-20 were synthesized (GenScript, Piscataway, NJ) and cloned into the pBSV-8His baculovirus expression vector, expressed in Spodoptera frugiperda Sf9 cells and purified by nickel affinity chromatography as described previously (42 (link), 43 (link)). FH SCRs 19-20 and mutant 19-20 fragments were expressed in E. coli (44 (link)).
Codon-optimized sequences of FHR-1, FHR-4B, FH SCRs 1-4, FH SCRs 8-14, FH SCRs 15-20 were synthesized (GenScript, Piscataway, NJ) and cloned into the pBSV-8His baculovirus expression vector, expressed in Spodoptera frugiperda Sf9 cells and purified by nickel affinity chromatography as described previously (42 (link), 43 (link)). FH SCRs 19-20 and mutant 19-20 fragments were expressed in E. coli (44 (link)).
5
Complement Pathway Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified human FH, factor B (FB), factor D (FD), properdin (factor P), C3b, FI, goat anti-human FH Ab, and goat anti-human FB Ab were obtained from Merck (Budapest, Hungary). The anti-FH mAb A254 was purchased from Quidel (Biomedica; Budapest, Hungary), and mAb OX24 was from Santa Cruz Biotechnology. HRP-conjugated goat anti-human C3 was purchased from MP Biomedicals (Solon, OH, USA). HRP-conjugated rabbit anti-goat Ig and HRP-conjugated goat anti-mouse Ig were from Dako (Hamburg, Germany). Bovine serum albumin (BSA) was from Applichem (Darmstadt, Germany). Normal human plasma was collected from healthy individuals after informed consent and pooled.
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
Revolutionizing how scientists
search and build protocols!