Can one Western blot protocol fit all antibodies?

Blot A: Anti-RbcS antibodies, made to a peptide derived from higher plant RbcS sequences (AS07 259)

Blot B: Anti-RbcS antibodies, made to a peptide conserved in algal and higher plant RbcS sequences (AS22 4825)

5 µg/well of total protein extracted freshly from Arabidopsis thaliana and Zea mays, denatured with 4xLDS at 70°C for 5 min. Proteins were separated on NuPAGE Bis-Tris SDS gel and blotted 1 h to Invitrogen PVDF (pore size of 0.2 µm), using wet transfer. Blot was blocked with 5% milk 4°C/ON with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 for 1 h/RT with agitation in TBS-T Blocking. The antibody solution was decanted, and the blot was rinsed briefly, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (AS09 607) diluted to 1:2500 in TBS-T Blocking for 30 min/RT with agitation. The blot was washed as above and developed for 2 min with Agrisera BCIP/NBT Plus.

Further optimization of the protocol is required for blot A: Protein load/well can be decreased to 2.5 µg, combined with primary antibody dilution increased to 1: 2000 1 h/RT incubation.

Each antibody-protein pair requires specific protocol optimization, and there is no one-protocol-fits-all solution.

You are welcome to use Agrisera Western blot resources or send us your results for consultation and help.

Blot A anti-RbcS antibody (AS07 259)
Lane:
1 - MW marker
2 - 5 µg of Arabidopsis thaliana whole leaf extract
3 - 5 µg of Zea mays whole leaf extract

Blot B anti-RbcS antibody (AS22 4825)
Lane:
1 - MW marker
2 - 5 µg of Arabidopsis thaliana whole leaf extract
3 - 5 µg of Zea mays whole leaf extract

Can one Western blot protocol fit all antibodies?

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