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



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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 Immunogen affinity purified serum in PBS pH 7.4.
Format Lyophilized
Quantity 50 ĩg
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 the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
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 highest sensitivity detection reagent. 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 highest sensitivity detection reagent. 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.

Related products

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

immunogen:

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


Reconstitution: - for reconstitution add 50 ĩl of sterile water
Host: Rabbit
Clonality: Polyclonal
Purity: Immunogen affinity purified serum in PBS pH 7.4.
Format: Lyophilized
Quantity: 50 ĩg
storage: Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
tested applications: Western blot (WB)
recommended dilution: 1 : 1000 (WB)
calculated | apparent molecular mass [kDa]:

38.8 kDa

Confirmed reactivity: Arabidopsis thaliana (protoplasts)
not reactive in:

 

Picture (footer):

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 highest sensitivity detection reagent. 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 highest sensitivity detection reagent. 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 (application): Reactivity on other tissues of Arabidopsis thaliana has not been confirmed as yet.
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.

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