showForm anti-MKK2 | Mitogen-activated protein kinase kinase 2 antibody

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MKK2 | Mitogen-activated protein kinase kinase 2

AS15 2905 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana (protoplasts)

MKK2 | Mitogen-activated protein kinase kinase 2 in the group Antibodies for Plant/Algal  / Developmental Biology / Signal transduction at Agrisera AB (Antibodies for research) (AS15 2905)

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Product Information

Immunogen

KLH-conjugated synthetic peptide derived from Arabidopsis thaliana sequence of MKK2, UniProt: Q9S7U9, TAIR: AT4G29810


Host Rabbit
Clonality Polyclonal
Purity Affinity purified serum in PBS, pH 7.4
Format Lyophilized
Quantity 50 µg
Reconstitution - for reconstitution add 50 ĩl of sterile water.
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications Western blot (WB)
Recommended dilution 1 : 1000 (WB)
Expected | apparent MW

38.8 kDa

Reactivity

Confirmed reactivity Arabidopsis thaliana (protoplasts)
Not reactive in

 

Application examples

Application examples

Application example


western blot using anti-MKK2 antibodies


Arabidopsis thaliana mesophyll protoplasts (Col-0) were isolated and transformed according to Yoo et al. (2007). 300 µl of protoplasts (2*105 protoplasts/ml) expressing MKK2/MKK4/MKK5-HA (or untransformed protoplasts as control) were pelleted by centrifugation. The resulting pellets were resuspended in standard Laemmli sample buffer and then the samples were denatured at 95°C for 5 min. Samples were separated on 10% SDS-PAGE and blotted 1 h onto NCL membrane using semi-dry transfer. The blot was blocked with 5% skimmed milk in TBS-T for 1h at room temperature (RT) with agitation. The blot was then incubated with the primary antibody (anti-MKK2) at a dilution of 1:1000 for 1 h at RT with agitation in 3% skimmed milk in TBS-T. The antibody solution was decanted and the blot was washed 4 times for 10 min with TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, Agrisera) diluted to 1:15000 in 3% skimmed milk in TBS-T for 1h at RT with agitation. The blot was washed as above and incubated for 5 min with Amersham ECL Prime Kit (GE Healthcare). Arrow indicates endogenous MKK2, star indicates MKK2-HA. Exposure time was 2 minutes. As a control, the blot was re-probed with anti-HA after stripping.

Courtesy Dr. Lennart Eschen-Lippold, Leibniz Institute of Plant Biochemistry, Germany western blot using anti-MKK2 Arabidopsis antibodies

Optical densities (OD600) of IPTG-induced E. coli expressing GST-MKK2/MKK4/MKK5 were determined and sample volumes based on the equation 1/OD600 were centrifuged to pellet bacteria. The resulting pellets were resuspended in 7 M urea, mixed with standard Laemmli sample buffer and then the samples were denatured at 95°C for 5 min. Samples were separated on 10% SDS-PAGE and blotted 1 h onto NCL membrane using semi-dry transfer. The blot was blocked with 5% skimmed milk in TBS-T for 1h at room temperature (RT) with agitation. The blot was then incubated with the primary antibody (anti-MKK2) at a dilution of 1:1000 for 1 h at RT with agitation in 3% skimmed milk in TBS-T. The antibody solution was decanted and the blot was washed 4 times for 10 min with TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, Agrisera) diluted to 1:15000 in 3% skimmed milk in TBS-T for 1h at RT with agitation. The blot was washed as above and incubated for 5 min with Amersham ECL Prime Kit (GE Healthcare). Exposure time was 2 minutes. As a control, the blot was re-probed with anti-GST after stripping.

Courtesy Dr. Lennart Eschen-Lippold, Leibniz Institute of Plant Biochemistry, Germany

Additional information

Reactivity on other tissues of Arabidopsis thaliana has not been confirmed as yet.

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Plant protein extraction buffer

Secondary antibodies

Background

Background MKK2 (Mitogen-activated protein kinase kinase 2) together with MKK2 and MKK4 function in a signaling pathway that modulates the expression of genes responding to biotic and abiotic stresses as well as pathogen defense by negative relugation of innate immunity.

Product citations

Selected references

To be added when available, antibody released in April 2016.