To confirm the expression of recombinant MVK (containing his-tag in N and C-terminal), expressed bacterial cell lysate was separated on 12% SDS-PAGE gel and probed with anti-his antibody at 1:1,000 dilution for 1h and then with anti-rabbit antibody (1:5,000 dilution; Jackson ImmunoResearch laboratories; AB_2307391) for 45 mins. For secretion experiments, culture supernatant of L. donovani was concentrated and run in SDS-PAGE. Secretory proteins were transferred onto PVDF membrane and processed for western blots analysis with anti-MVK antibody raised in rabbit at 1:1,000 dilutions for 1 h and then with anti-rabbit antibody (1:5,000 dilution; Jackson ImmunoResearch laboratories; AB_2307391) at 1:5,000 dilutions for 45 mins. Antibody antigen complexes were detected by enhanced chemiluminescence (GE Healthcare; #RPN2209) and bands were visualized using Image J software. Band densitometry was performed using ImageJ software. To validate that the supernatant containing secretory proteins was collected from equal number of cells, cell lysate was prepared for each condition and probed with anti-actin antibody which was kindly provided by Dr Anuradha Dube.
Anti his antibody
Manufactured by Jackson ImmunoResearch
Anti-his antibody is a laboratory reagent used to detect and purify proteins that have been engineered to contain a histidine (His) tag. The antibody is specific to the His tag, allowing for the identification and isolation of tagged proteins from complex mixtures.
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Lab products found in correlation
2 protocols using anti his antibody
1
Validating Recombinant MVK Expression
2
Arabidopsis and Rice Growth Conditions
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Growth conditions for Arabidopsis and rice Nipponbare (Oryza sativa L. ssp. japonica) were described previously (Yang et al., 2012; Qi et al., 2015a Qi et al., , 2015b;; Huang et al., 2017) . Coronatine (C8115), jasmonic acid (J2500), MeJA (S4400), and IAA (I2886) were purchased from Sigma. Anti-Biotin antibody and anti-His antibody were purchased from Jackson ImmunoResearch and Sigma, respectively.
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