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 rabbit ig
Manufactured by Agilent Technologies
Sourced in Germany
HRP conjugated goat anti-rabbit Ig is a secondary antibody used in various immunoassay techniques. It consists of goat-derived antibodies against rabbit immunoglobulins, conjugated with horseradish peroxidase (HRP) enzyme. This product can be used to detect and quantify the presence of rabbit primary antibodies in samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse 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)
Complete protease inhibitor tablet, by Roche (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Bapta am, by Merck Group (1 mentions)
Acutase, by Merck Group (1 mentions)
Pe conjugated anti cd14, by BD (1 mentions)
Apc conjugated anti cd4, by BD (1 mentions)
Heparin, by Leo pharma (1 mentions)
Normal goat igg, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated rabbit anti mouse ig, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Gammabind g sepharose, by GE Healthcare (1 mentions)
5 protocols using hrp conjugated goat anti rabbit ig
1
Western Blotting of Protein Samples
2
Recombinant SOD3 Protein Production and Antibody Development
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Human serum, M-CSF, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), acutase, and ovalbumin was from Sigma. Heparin (5000 U/ml) was obtained from Leo Pharma, Denmark and complete protease inhibitor tablets was from Roche.
Proteins: Recombinant wild-type SOD3 and SOD3 containing a C-terminal c-Myc tag (SOD3-cMyc) was produced as previously described [42 (link)]. For the generation of the SOD3-cMyc expression plasmid, we used the forward primer (5′-TATACAGCTAGCATGCTGGCGCTACTGTGTTCC-3′) and a reverse primer encompassing the cMyc tag (underlined) (5′-TATGAATTCTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCG GCGGCCTTGCACTCGCTCTC-3′) and cloned the product into the pIRES vector. The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose. Recombinant human and murine IFNγ was obtained from Invitrogen. ovalbumin was from Sigma. Human receptor-associated protein (RAP) was obtained from ENZO life sciences (BML-SE552). HRP-conjugated goat anti-rabbit Ig was from DAKO. Flow cytometric analyses were performed using PE-conjugated anti-CD14 (B&D Biosciences), APC-conjugated anti-CD4 (B&D Biosciences) or APC-conjugated mAb5G8D4 anti-SOD3.
Proteins: Recombinant wild-type SOD3 and SOD3 containing a C-terminal c-Myc tag (SOD3-cMyc) was produced as previously described [42 (link)]. For the generation of the SOD3-cMyc expression plasmid, we used the forward primer (5′-TATACAGCTAGCATGCTGGCGCTACTGTGTTCC-3′) and a reverse primer encompassing the cMyc tag (underlined) (5′-TATGAATTCT
3
Characterization of Extracellular Superoxide Dismutase
4
Purification and Expression of Complement Proteins
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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
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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.
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