RGA | DELLA protein RGA

AS11 1630 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana

Product Information

Immunogen

KLH-conjugated peptide chosen from RGA of Arabidopsis thaliana UniProt: Q9SLH3,TAIR: At2g01570

Host Rabbit

Clonality Polyclonal

Purity Immunogen affinity purified serum in PBS pH 7.4.

Format Lyophilized

Quantity 50 µg

Reconstitution For reconstitution add 50 µl 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

64 | 64 kDa

Reactivity

Confirmed reactivity Arabidopsis thaliana

Predicted reactivity Arabidopsis thaliana

Not reactive in

Brassica napus, Populus sp., Rosa chinensis, Triticum aestivum

Application examples

Application example

40 µg of total protein from 5-d-old dark-grown Arabidopsis thaliana seedlings extracted with 50 mM Tris-HCl pH 7.5,, 10% glycerol, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF, and 1x protease inhibitor cocktail (Roche) were separated on 4-20 % SDS-PAGE and blotted 1h to PVDF. m: mock, P: paclobutyrazol, P+G: PAC+GAs. Blots were blocked with 2% blocking reagent (GE Healthcare) in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602 ) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescence detection reagent, according to the manufacturers instructions. Exposure time was 20 seconds in a LAS-3000 Imager (Fuji).

Courtesy of Dr. David Alabadi, IBMCP (CSIC-UPV), Valencia, Spain

westrn blot using anti-RGA antibodies

Arabidopsis thaliana seedlings were ground in liquid nitrogen (100 µl of 2.5 x Laemli for 80-120 mg of homogenized material) and boiled in 2,5x Laemmli Buffer (WITH 60 Mm DTT final concentration) otherwise RGA protein will degrade. Plants were grown on 1/ MS for 15 days and were treated with 1 µM GA for 2 hours (GA+) or without hormone (GA-). Total protein extracts were denatured for 2 min. at 95°C and were separated on 10% SDS-PAGE and blotted overnight to PVDF using tank transfer. Blots were blocked for 1.5 h with TBS-T containing 5% low fat milk for 2h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h 30min at RT with agitation. The antibody solution was decanted and the blot was washed 5 x 10min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:10 000 for 1h 30 min at RT with agitation. The blot was washed 6 x 10min in TBS-T at RT with agitation and developed for 5 min with ECL according to the manufacturer's instructions. Exposure time was 5 seconds with West Femto (Pierce).

Courtesy of Kamila Jaronczyk, IBB, Warsaw, Poland

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 30149837

Journal: Elife

Figure Number: 2C

Published Date: 2018-08-28

First Author: Chahtane, H., Nogueira Füller, T., et al.

Impact Factor: 7.448

Open Publication

RGL2, GAI, RGA and ABI5 antibodies specificity.Protein gel blot analysis using antibodies to RGL2 (A), ABI5 (B), GAI and RGA (C) as indicated. Protein extracts from Col-0 (WT), rgl2-13 (rgl2), rgl2-SK54 rga-28 (rgl2 rga), rgl2-SK54 rga-28 gai-t6 (rgl2 rga gai) and abi5-3 (abi5) mutant seeds harvested after seed imbibition in presence or absence of PAC (PAC) as indicated. UGPase protein levels are used as a loading control.

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 30149837

Journal: Elife

Figure Number: 6A

Published Date: 2018-08-28

First Author: Chahtane, H., Nogueira Füller, T., et al.

Impact Factor: 7.448

Open Publication

AMB does not interfere with GA-dependent GAI and RGA protein downregulation.WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high DELLA accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGA (A) and GAI (B) protein levels are followed by protein gel blot analysis over the time points indicated in red as indicated. UGPase protein levels are used as a loading control.

Reactant: Arabidopsis thaliana (Thale cress)

Application: Western Blotting

Pudmed ID: 30149837

Journal: Elife

Figure Number: 6B

Published Date: 2018-08-28

First Author: Chahtane, H., Nogueira Füller, T., et al.

Impact Factor: 7.448

Open Publication

AMB and PAC induce different changes on DELLA protein accumulation.AMB promotes DELLA factor activity to stimulate ABA signaling. (A-C) Protein gel blot analysis using antibodies to RGL2 (A), RGA (B) and GAI (C) as indicated. Protein extracts from WT (Col seeds harvested 24 hr after seed imbibition in absence (MS) or presence of 10 µM PAC (PAC), 5 µM ABA (ABA) or 50 µM AMB (AMB). UGPase protein levels are used as a loading control. (D) AMB does not interfere with GA-dependent RGL2 protein downregulation. WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high RGL2 accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGL2 protein levels are followed by protein gel blot analysis over the time points indicated in red. UGPase protein levels are used as a loading control. Germination percentage at each time point is indicated. The asterisk (*) represents and unspecific banc detected by the RGL2 antibody. (E) AMB stimulates DELLA activity to promote ABA-dependent responses. Histograms show RGL2, ABI5, EM1 and NCED6 mRNA accumulation in WT (Col) and ?della seeds treated as described in A. For each time point, mRNA levels are normalized to mRNA levels in WT seeds sown in absence of AMB. Data represent mean ±standard deviation (three replicates).AMB does not interfere with GA-dependent GAI and RGA protein downregulation.WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high DELLA accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGA (A) and GAI (B) protein levels are followed by protein gel blot analysis over the time points indicated in red as indicated. UGPase protein levels are used as a loading control.

Additional information

RGA protein is prone to degradation therefore caution has to be taken during protein extraction

Background

Background DELLA protein RGA is a putative transcriptional regulator that acts as a repressor of the gibberellin (GA) signaling pathway. It is a member of VHIID/DELLA regulatory family and is the most sensitive to GA application, compare to other DELLA proteins.Involved in fruit and flower development. Synonymes: GAI-related sequence, GRAS family protein 10, AtGRAS-10, Repressor on the ga1-3 mutant, Restoration of growth on ammonia protein 1.

Product citations

Selected references Dong et al. (2021). An HB40 - Jungbrunnen1 - GA 2-OXIDASE regulatory module for gibberellin homeostasis in Arabidopsis
Yan et al. (2020). FKF1 F-box Protein Promotes Flowering in Part by Negatively Regulating DELLA Protein Stability Under Long-Day Photoperiod in Arabidopsis. J Integr Plant Biol . 2020 May 19. doi: 10.1111/jipb.12971.
Ferrero et al. (2019). Class I TCP transcription factors target the gibberellin biosynthesis gene GA20ox1 and the growth promoting genes HBI1 and PRE6 during thermomorphogenic growth in Arabidopsis. Plant Cell Physiol. 2019 Jul 11. pii: pcz137. doi: 10.1093/pcp/pcz137.
Lorrai et al. (2018). Abscisic acid inhibits hypocotyl elongation acting on gibberellins, DELLA proteins and auxin. AoB Plants. 2018 Oct 5;10(5):ply061. doi: 10.1093/aobpla/ply061.
Chahtane et al. (2018). The plant pathogen Pseudomonas aeruginosa triggers a DELLA-dependent seed germination arrest in Arabidopsis. Elife. 2018 Aug 28;7. pii: e37082. doi: 10.7554/eLife.37082.
Shahnejat-Bushehri et al. (2016). Arabidopsis NAC transcription factor JUB1 regulates GA/BR metabolism and signalling. NATURE PLANTS 2: Article number: 16013, 2016.
Crocco et al. (2015). The transcriptional regulator BBX24 impairs DELLA activity to promote shade avoidance in Arabidopsis thaliana. Nat Commun. 2015 Feb 6;6:6202. doi: 10.1038/ncomms7202.
Leone et al. (2014). To grow or defend? Low red : far-red ratios reduce jasmonate sensitivity in Arabidopsis seedlings by promoting DELLA degradation and increasing JAZ10 stability. New Phytol. 2014 Oct;204(2):355-67. doi: 10.1111/nph.12971. Epub 2014 Aug 7.

All references:	Dong et al. (2021). An HB40 - Jungbrunnen1 - GA 2-OXIDASE regulatory module for gibberellin homeostasis in Arabidopsis Yan et al. (2020). FKF1 F-box Protein Promotes Flowering in Part by Negatively Regulating DELLA Protein Stability Under Long-Day Photoperiod in Arabidopsis. J Integr Plant Biol . 2020 May 19. doi: 10.1111/jipb.12971. Ferrero et al. (2019). Class I TCP transcription factors target the gibberellin biosynthesis gene GA20ox1 and the growth promoting genes HBI1 and PRE6 during thermomorphogenic growth in Arabidopsis. Plant Cell Physiol. 2019 Jul 11. pii: pcz137. doi: 10.1093/pcp/pcz137. Lorrai et al. (2018). Abscisic acid inhibits hypocotyl elongation acting on gibberellins, DELLA proteins and auxin. AoB Plants. 2018 Oct 5;10(5):ply061. doi: 10.1093/aobpla/ply061. Chahtane et al. (2018). The plant pathogen Pseudomonas aeruginosa triggers a DELLA-dependent seed germination arrest in Arabidopsis. Elife. 2018 Aug 28;7. pii: e37082. doi: 10.7554/eLife.37082. Shahnejat-Bushehri et al. (2016). Arabidopsis NAC transcription factor JUB1 regulates GA/BR metabolism and signalling. NATURE PLANTS 2: Article number: 16013, 2016. Crocco et al. (2015). The transcriptional regulator BBX24 impairs DELLA activity to promote shade avoidance in Arabidopsis thaliana. Nat Commun. 2015 Feb 6;6:6202. doi: 10.1038/ncomms7202. Leone et al. (2014). To grow or defend? Low red : far-red ratios reduce jasmonate sensitivity in Arabidopsis seedlings by promoting DELLA degradation and increasing JAZ10 stability. New Phytol. 2014 Oct;204(2):355-67. doi: 10.1111/nph.12971. Epub 2014 Aug 7.
More images:	Reactant: Arabidopsis thaliana (Thale cress) Application: Western Blotting Pudmed ID: 30149837 Journal: Elife Figure Number: 2C Published Date: 2018-08-28 First Author: Chahtane, H., Nogueira Füller, T., et al. Impact Factor: 7.448 Open Publication RGL2, GAI, RGA and ABI5 antibodies specificity.Protein gel blot analysis using antibodies to RGL2 (A), ABI5 (B), GAI and RGA (C) as indicated. Protein extracts from Col-0 (WT), rgl2-13 (rgl2), rgl2-SK54 rga-28 (rgl2 rga), rgl2-SK54 rga-28 gai-t6 (rgl2 rga gai) and abi5-3 (abi5) mutant seeds harvested after seed imbibition in presence or absence of PAC (PAC) as indicated. UGPase protein levels are used as a loading control. Reactant: Arabidopsis thaliana (Thale cress) Application: Western Blotting Pudmed ID: 30149837 Journal: Elife Figure Number: 6A Published Date: 2018-08-28 First Author: Chahtane, H., Nogueira Füller, T., et al. Impact Factor: 7.448 Open Publication AMB does not interfere with GA-dependent GAI and RGA protein downregulation.WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high DELLA accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGA (A) and GAI (B) protein levels are followed by protein gel blot analysis over the time points indicated in red as indicated. UGPase protein levels are used as a loading control. Reactant: Arabidopsis thaliana (Thale cress) Application: Western Blotting Pudmed ID: 30149837 Journal: Elife Figure Number: 6B Published Date: 2018-08-28 First Author: Chahtane, H., Nogueira Füller, T., et al. Impact Factor: 7.448 Open Publication AMB and PAC induce different changes on DELLA protein accumulation.AMB promotes DELLA factor activity to stimulate ABA signaling. (A-C) Protein gel blot analysis using antibodies to RGL2 (A), RGA (B) and GAI (C) as indicated. Protein extracts from WT (Col seeds harvested 24 hr after seed imbibition in absence (MS) or presence of 10 µM PAC (PAC), 5 µM ABA (ABA) or 50 µM AMB (AMB). UGPase protein levels are used as a loading control. (D) AMB does not interfere with GA-dependent RGL2 protein downregulation. WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high RGL2 accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGL2 protein levels are followed by protein gel blot analysis over the time points indicated in red. UGPase protein levels are used as a loading control. Germination percentage at each time point is indicated. The asterisk (*) represents and unspecific banc detected by the RGL2 antibody. (E) AMB stimulates DELLA activity to promote ABA-dependent responses. Histograms show RGL2, ABI5, EM1 and NCED6 mRNA accumulation in WT (Col) and ?della seeds treated as described in A. For each time point, mRNA levels are normalized to mRNA levels in WT seeds sown in absence of AMB. Data represent mean ±standard deviation (three replicates).AMB does not interfere with GA-dependent GAI and RGA protein downregulation.WT seeds are imbibed in presence of 5 µM PAC for 30 hr to trigger high DELLA accumulation. Thereafter, seeds are transferred to germination plates containing 5 µM PAC or containing 5 µM PAC and 50 µM AMB. After 12 hr, 1 µM GA is further added and RGA (A) and GAI (B) protein levels are followed by protein gel blot analysis over the time points indicated in red as indicated. UGPase protein levels are used as a loading control.
Picture (footer):	Application example 40 µg of total protein from 5-d-old dark-grown Arabidopsis thaliana seedlings extracted with 50 mM Tris-HCl pH 7.5,, 10% glycerol, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF, and 1x protease inhibitor cocktail (Roche) were separated on 4-20 % SDS-PAGE and blotted 1h to PVDF. m: mock, P: paclobutyrazol, P+G: PAC+GAs. Blots were blocked with 2% blocking reagent (GE Healthcare) in TBS-T for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602 ) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with chemiluminescence detection reagent, according to the manufacturers instructions. Exposure time was 20 seconds in a LAS-3000 Imager (Fuji). Courtesy of Dr. David Alabadi, IBMCP (CSIC-UPV), Valencia, Spain Arabidopsis thaliana seedlings were ground in liquid nitrogen (100 µl of 2.5 x Laemli for 80-120 mg of homogenized material) and boiled in 2,5x Laemmli Buffer (WITH 60 Mm DTT final concentration) otherwise RGA protein will degrade. Plants were grown on 1/ MS for 15 days and were treated with 1 µM GA for 2 hours (GA+) or without hormone (GA-). Total protein extracts were denatured for 2 min. at 95°C and were separated on 10% SDS-PAGE and blotted overnight to PVDF using tank transfer. Blots were blocked for 1.5 h with TBS-T containing 5% low fat milk for 2h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h 30min at RT with agitation. The antibody solution was decanted and the blot was washed 5 x 10min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:10 000 for 1h 30 min at RT with agitation. The blot was washed 6 x 10min in TBS-T at RT with agitation and developed for 5 min with ECL according to the manufacturer's instructions. Exposure time was 5 seconds with West Femto (Pierce). Courtesy of Kamila Jaronczyk, IBB, Warsaw, Poland
additional information (application):	RGA protein is prone to degradation therefore caution has to be taken during protein extraction
Confirmed reactivity:	Arabidopsis thaliana
predicted reactivity:	Arabidopsis thaliana
not reactive in:	Brassica napus, Populus sp., Rosa chinensis, Triticum aestivum
calculated \| apparent molecular mass [kDa]:	64 \| 64 kDa
Clonality:	Polyclonal
Format:	Lyophilized
Host:	Rabbit
immunogen:	KLH-conjugated peptide chosen from RGA of Arabidopsis thaliana UniProt: Q9SLH3,TAIR: At2g01570
Purity:	Immunogen affinity purified serum in PBS pH 7.4.
Quantity:	50 µg
recommended dilution:	1 : 1000 (WB)
Reconstitution:	For reconstitution add 50 µl 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)
background:	DELLA protein RGA is a putative transcriptional regulator that acts as a repressor of the gibberellin (GA) signaling pathway. It is a member of VHIID/DELLA regulatory family and is the most sensitive to GA application, compare to other DELLA proteins.Involved in fruit and flower development. Synonymes: GAI-related sequence, GRAS family protein 10, AtGRAS-10, Repressor on the ga1-3 mutant, Restoration of growth on ammonia protein 1.

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