Western blots were performed as previously described [37 (link)]. Briefly, ST764 isolates (six randomly selected isolates each in t002 isolates and t1084 isolates, and these isolates same apply to the experiment that follows) were grown in TSB with shaking for 24 h and bacterial culture supernatants were collected. The protein concentration of each sample was determined by Bradford assays to keep the total amount of protein loaded for SDS-PAGE consistent in the twelve isolates. Proteins were mixed with Omni-EasyTM Protein Sample Loading Buffer (EpiZyme Biotechnology Co., Ltd., Shanghai, China) and heated for 10 min at 95°C. Samples were separated by 12.5% SDS-PAGE and were transferred to the PVDF membrane. After blocking with 5% bovine serum albumin (BSA) at 4°C for overnight, the membrane was incubated with a rabbit anti-Hla IgG antibody (Sigma) at a 1/2500 dilution. The membrane was then incubated with Goat Anti-Rabbit IgG HRP (Biosharp) at a 1/5000 dilution. Omni-ECLTM Pico Light Chemiluminescence Kit (EpiZyme Biotechnology Co., Ltd., Shanghai, China) was used to develop image.
Goat anti rabbit igg hrp
Manufactured by Biosharp
Sourced in China, Estonia
Goat anti-rabbit IgG-HRP is a secondary antibody that binds to rabbit primary antibodies. It is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for signal amplification in various immunoassays and detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg hrp, by Biosharp (3 mentions)
Rabbit anti hla igg antibody, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Bcl 2, by Proteintech (1 mentions)
Bl001a, by Biosharp (1 mentions)
Sds page loading buffer, by Meilun (1 mentions)
Ap0475, by ABclonal (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Biosharp (1 mentions)
Ab195352, by Abcam (1 mentions)
Ab104224, by Abcam (1 mentions)
Ab195377, by Abcam (1 mentions)
Ab92821, by Abcam (1 mentions)
Dylight 488 donkey anti mouse igg, by EarthOx (1 mentions)
Ab32503, by Abcam (1 mentions)
Dylight 594 donkey anti rabbit igg, by EarthOx (1 mentions)
Ap0060, by Bioworld Technology (1 mentions)
Af6139, by Affinity Biosciences (1 mentions)
6 protocols using goat anti rabbit igg hrp
1
Western Blot Analysis of Staphylococcus aureus Hla
2
Modulation of Autophagy and Apoptosis
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After 24 h of aloin, chloroquine, rapamycin and starvation administration, HOS and MG-63 cell lines were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. Protein samples of same amount were separated by western blot. Blot intensity was analyzed by Image J (v1.51). The primary antibodies applied in this research were as follows: AKT, ATG5, Beclin1, Bcl-2, bcl-xl, cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, cleaved PARP, mTOR, pho-mTOR (SER2448), pho-AKT (SER473), PI3Kα (p110), SQSTM1 (P62), LC3B. Beta-actin was used as internal reference. The secondary antibodies were as follows: goat anti-rabbit IgG–HRP (1:2000, Biosharp) and goat anti-mouse IgG–HRP (1:2000, Biosharp).
3
Apoptosis and Metastasis Inhibition Assay
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Roswell Park Memorial Institute (RPMI)-1640 was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Sijiqing (Hangzhou, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO, USA). An annexin V/PI staining kit was purchased from BestBio Tech. Co., Shanghai, China. The specific primary antibodies against Akt, Bax, Bcl-2, and β-actin were purchased from Proteintech (Rosemont, IL, USA) and antibodies against HIF-1α, TIMP1, MMP-2, and MMP-9 were purchased from Abcam (Cambridge, MA, USA). The secondary antibodies used in this study were goat anti-rabbit IgG-HRP or anti-mouse IgGHRP (BioSharp, Hefei, China).
4
Western Blot Analysis of Protein Extracts
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Total protein was extracted from cells and tissues using RIPA lysis buffer (#P0013B, Beyotime, Beijing, China). Protein concentrations were quantified using the BCA protein assay kit (#BL521A, Biosharp, Beijing, China). Protein samples were mixed with SDS–PAGE loading buffer (#MB2479, Meilunbio, Dalian, China), separated on a SDS-PAGE gel, and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with primary antibodies against TNFα (26405-1-AP, 1:2000, Proteintech), phosphorylated P65 (p-P65) (AP0475, 1:2000, ABCLONAL, Woburn, MA, USA), total P65 (66535-1-Ig, 1:2000, Proteintech), and GAPDH (10494-1-AP, 1:5000, Proteintech). Next, the membranes were incubated with goat anti-rabbit IgG-HRP (BL003A, 1:5000, Biosharp) or goat anti-mouse IgG-HRP (BL001A, 1:5000, Biosharp) secondary antibodies. ECL color exposure was used to detect immunoreactive bands. An Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA) was used to quantify protein bands, using GAPDH as the internal reference.
5
Antibody Panel for m6A RNA Modifications
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The following antibodies were used: anti-m6A antibody (202003, Synaptic Systems, 1:1000 for Dot blot, 1:100 for Immunofluorescence), Mouse Anti-NeuN antibody (ab104224, Abcam,1:500) rabbit anti-ALKBH5 antibody (ab195377, Abcam, 1:1000), rabbit anti-METTL3 antibody (ab195352, Abcam, 1:1000), mouse monoclonal to FTO (ab92821, Abcam, 1:1000), BCL-2 antibody (AF6139, Affinity, 1:1000), rabbit anti-Bax antibody (ab32503, Abcam, 1:5000), Akt (4691T, Cell Signaling Technology, 1:1000), Phospho-Akt-Ser473 (4060S, Cell Signaling Technology, 1:1000), mTOR (2983T, Cell Signaling Technology, 1:1000), Phospho-mTOR-Ser2448 (5536T, Cell Signaling Technology, 1:1000), PARP (9542T, Cell Signaling Technology, 1:1000), β-Actin polyclonal antibody (AP0060, Bioworld Technology, 1:1000), Goat anti-rabbit IgG-HRP (BL003A, Biosharp Life Sciences, 1:5000), Goat anti-mouse IgG-HRP(BL001A, Biosharp Life Sciences, 1:5000), Dylight 594-donkey anti-rabbit IgG (E032421-01, Earthox, 1:500), Dylight 488-donkey anti-mouse IgG (E032211-01, Earthox, 1:500). Other reagents information is listed in Supplementary Materials .
6
Western Blot Analysis of NF-κB Regulators
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Protein isolation, concentration quantification and separation, as well as protein were transferred onto PVDF membranes (Millipore, Bedford, MA, USA), blocked with 5% fat-free milk, and membrane incubation was performed as described previously [21 (link)]. The primary antibodies included an anti-BCL-3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-32741), an anti-IκBα antibody (Santa Cruz Biotechnology, sc-1643), an anti-IκBβ antibody (Santa Cruz Biotechnology, sc-390622), an anti-IκBεantibody (Santa Cruz Biotechnology, sc-7275), an anti-IKKγ antibody (Santa Cruz Biotechnology, sc-166398), an anti-NFKBID antibody (Abcam, Cambridge, UK, ab232913), and an anti-IκBζ antibody (Abcam, ab155142). The antibodies were validated by ovine proteins, and suitable for sheep. Secondary antibody goat anti-mouse IgG-horseradish peroxidase-conjugated (HRP) (Biosharp, Tallinn, Estonia, BL001A) or goat anti-rabbit IgG-HRP (Biosharp, BL003A) was incubated in a 1:10 000 dilution. GAPDH (anti-GAPDH antibody, Santa Cruz Biotechnology, sc-20357, 1:1000) was used as the loading control. An enhanced chemiluminescence kit (Tiangen Biotech Co., Ltd., Beijing, China) was used to detect target proteins. The band intensity was quantified with Quantity One V452 software (Bio-Rad Laboratories, Hercules, CA, USA).
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