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Thank you for participating in Agrisera Antibody Testing Program!
- 20 ug protein load/well
- Blocking: 5 % non-fat milk 1h/RT
- Primary antibodies: 1: 1000 ON/4C TBS-T
- Secondary antibodies (Agrisera): 1: 25 000 1h/RT
- Detection: AgriseraSuperBright (for proteins of low expression) or AgriseraBright (for proteins of moderate expression)
Results are to be returned in the following format:
Description of each lane (as in the example below):
1 - 50 ug of Arabidopsis thaliana whole leaf extract
2 - 50 ug of Arabidopsis thaliana mutant
Mark: MW markers: in example below: MagicMark from Invitrogen
Mark MW of a target protein to be detected
Provide an IMAGE (whole membrane with MW markers), of a good quality, like in the example below:
Fill in ALL experimental details in the template below:
µg/well of total protein extracted freshly from with write exact buffer components ..... and denatured with....at °C or 70°C for 2-5 min.... were separated on …. % SDS-PAGE and blotted 1h to PVDF/nitrocellulose (pore size of um), using wet, semi-dry or dry transfer. Blot was blocked with % milk or % BSA for 1h/RT or 4°C/ON with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h/RT with agitation in TBS-T or ON/4°C 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 Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25 000 in for 1h/RT with agitation. The blot was washed as above and developed for min with Agrisera ECLBright or AgriseraECLSuperBright with name and supplier. Exposure time was.....seconds.
Questions to answer:
Was used material freshly extracted or stored?
Was a blocking reagent prepared freshly?
How was primary antibody stored?
Was primary antibody or any other reagent re-used?
Was incubation volume for each step kept the same?
Thank you for your contribution!
In return we offer a 10 % discount on your next order from Agrisera