Cells were lysed in M2 Buffer (20 mM Tris HCl, pH 7.6, 0.5% NP40, 250 mM NaCl, 3 mM EDTA and 3 mM EGTA), supplemented with 1×EDTA-free protease inhibitor cocktail (MedChemExpress, HY-K0010). Protein concentration was quantified by the Bradford reagent (BIO-RAD, 500-0205). Protein samples were mixed with Laemmli loading buffer and boiled for 10 minutes. Subsequently, samples were separated by SDS polyacrylamide gelelectrophoresis (SDS-PAGE), then were transferred to polyvinylidene fluoride (PVDF) membranes (PALL, BSP0161). Blots were incubated with primary antibodies overnight at 4°C followed by HRP-conjugated species-specific secondary antibodies (Jackson Immuno Research, 1:5000) at room temperature for one hour. Finally, the signals were detected by ECL luminescence reagent (Absin, abs920). The following antibodies were used at the indicated dilutions: WDR12 (1:1000, NBP1-53111, Novus), Pes1 (1:1000, b88543, Abcam), Bop1 (1:1000, 28366-1-AP, Proteintech), SOX2 (1:1000, MAB4423, Millipore), Olig2 (1:1000, sc-48817, Santa Cruz), GFAP (1:1000, Z0344, Dako), GAPDH (1:1000, 3683s, Cell Signaling).
Species specific hrp conjugated secondary antibodies
Manufactured by Jackson ImmunoResearch
Species-specific HRP-conjugated secondary antibodies are laboratory tools designed to detect and visualize target proteins or molecules in various immunoassays. These antibodies are conjugated with the enzyme Horseradish Peroxidase (HRP), which can catalyze a color-producing reaction when exposed to a suitable substrate. The species-specificity ensures the antibodies recognize and bind to the appropriate primary antibodies, enabling the detection of the target of interest.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay, by Bio-Rad (3 mentions)
Myecl imager, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Z0344, by Agilent Technologies (1 mentions)
Mab4423, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Pmsf complete, by Roche (1 mentions)
Gapdh, by Abcam (1 mentions)
Hybond nitrocellulose membrane, by Cytiva (1 mentions)
Gapdh, by SCBT (1 mentions)
Hpv16 e7, by Santa Cruz Biotechnology (1 mentions)
Hpv16 e6, by GeneTex (1 mentions)
Hpv18 e6, by Santa Cruz Biotechnology (1 mentions)
Hpv18 e7, by Abcam (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ab81321, by Abcam (1 mentions)
Ab23393, by Abcam (1 mentions)
6 protocols using species specific hrp conjugated secondary antibodies
1
Protein Lysate Preparation and Western Blot Analysis
2
Western Blot Analysis of PW1 and GAPDH in Brain Homogenates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated brains were homogenized in lysis buffer (150mM NaCl, 50mM Hepes pH7.6, 1% NP-40, 0,5% sodium deoxycholate, 5mM EDTA) supplemented with 1mM PMSF, Complete (Roche), 20mM NaF, 10mM b-glycerophosphate, 5mM Na-pyrophosphate, and 1mM Na-orthovanadate. Equal amounts of protein were separated by electrophoresis (Novex NuPAGE Bis-Tris protein gel 4–12% or 5% home-made Bis-Tris gel) and transferred to a PVDF membrane in 20% methanol transfer buffer. Membranes were probed with polyclonal rabbit antibodies to PW1 (rabbit, 1:10,000) (Relaix et al., 1996) and GAPDH (Abcam). Antibody binding was visualized using horse-radish peroxidase (HRP)-conjugated species-specific secondary antibodies (Jackson ImmunoResearch) followed by enhanced chemiluminescence (Pierce).
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto Hybond nitrocellulose membrane (Amersham biosciences), followed by immunoblot analysis. Antibodies used in this study were as follows: CREB1 (9197, CST), phospho‐CREB1 (9198, CST), HPV16 E6 (GTX132686, GeneTex), HPV16 E7 (sc‐65711, Santa Cruz), HPV18 E6 (sc‐365089, Santa Cruz), HPV18 E7 (ab100953, Abcam), Snail (3879, CST), Slug (9585, CST), GAPDH (G‐9, SCBT), GFP (sc‐9996, SCBT), and FLAG (F3165, Sigma). Western blots were visualized with species‐specific HRP conjugated secondary antibodies (Jackson ImmunoResearch) and ECL (Thermo/Pierce).
4
Western Blot Analysis of Nerve Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Muscle and sciatic nerve (SN) tissues of both sexes were excised and homogenized in lysis buffer containing PBS, 1% Triton X-100 (Sigma-Aldrich), and 1× protease inhibitors (Roche Diagnostics), followed by centrifugation and collection of the supernatant. Protein concentration was determined using the Bio-Rad Protein Assay. Protein samples were denatured by boiling in SDS sample buffer, which were then electrophoresed in 10% polyacrylamide gels (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane and then immunoblotted with appropriate primary antibodies: Sema3A (Abcam, ab23393; 1:1000); NRP1 (Abcam, ab81321; 1:1000); Sema3B (Abcam, ab48197; 1:2000); NRP2 (Cell Signaling D39A5, 1: 1000); GFP (Abcam ab13970; 1:5000); tubulin (1:10,000) and ERK (1:10,000), diluted in 5% (w/v) skim milk (BD Difco) in TBS-T, followed by species-specific HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; 1:10,000) and visualized using a myECL imager (Thermo Fisher Scientific), according to the manufacturer's instructions. Quantification was performed using ImageJ software.
5
Western Blot Analysis of Brain Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain tissue was excised and homogenized in lysis buffer containing PBS, 1% Triton X-100 (Sigma), and 1% protease inhibitors (Roche), followed by centrifugation and collection of the supernatant. Protein concentration was determined using the Bio-Rad Protein Assay. Protein samples were denatured by boiling in SDS sample buffer and then electrophoresed in 10% polyacrylamide gels (SDS-PAGE). Proteins were transferred to a nitrocellulose membrane and then immunoblotted with appropriate primary antibodies: Elavl2 1:1,000 (Rabbit; Proteintech, 14008-1-AP); Tubulin-α 1:10,000 (mouse; Abcam, ab7291) diluted in 5% (w/v) Skim-milk (BD Difco) in TBS-T, followed by species-specific HRP-conjugated secondary antibodies (Jackson Laboratories) and visualized using a myECL imager (Thermo), according to the manufacturer’s instructions. Quantification was performed using ImageJ software.
6
Sciatic Nerve Axoplasm Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sciatic nerve axoplasm was isolated by excising and cutting sciatic nerves into short segments, followed by detergent‐free buffer homogenized with PBS X1 protease and phosphatase inhibitors (Roche), followed by centrifugation and collection of the supernatant. Complete sciatic nerve extracts were achieved in the same manner with the exception of adding 1% Triton X‐100. The protein concentration was determined using the Bio‐Rad Protein Assay. Protein samples were denatured by boiling in SDS sample buffer and then electrophoresed in 8% polyacrylamide gels (SDS–PAGE). Proteins were transferred to a nitrocellulose membrane and then immunoblotted with appropriate primary antibodies: anti‐CRMP4—1:2,000 (Millipore AB5454), anti‐DIC—1:1,000 (Millipore MAB1618), anti‐p150 1:250 (BD Bioscience 611003), anti‐Flag 1:4,000 (Sigma‐Aldrich F3165), anti‐Tubulin 1:10,000 (ab7291), and anti‐tERK 1:10,000 (M5670), diluted in 5% (w/v) skim milk (BD Difco) in TBS‐T, followed by species‐specific HRP‐conjugated secondary antibodies (Jackson Laboratories) and visualized using a myECL imager (Thermo), according to the manufacturer’s instructions. ImageJ software was used for quantification.
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!