Application
Western blot
Sample
Mouse Cell lysate - whole cell (CH12-F3 cell line)
Gel Running Conditions
Reduced Denaturing (12)
Loading amount
40 µg
Treatment
One sample was treated with cytokine cocktail CIT to induce AID expression (see additional notes for more information).
Specification
CH12-F3 cell line
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBS-5% BSA-0.1% Tween-20
Concentration
1 µg/ml
Secondary antibody
Dilution
1/10000
Name
Non-Abcam antibody was used: Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 680
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 680
Detection
Detection method
Fluorescence
Negative control
CH12F3 Cells AID knock-out
Exposure
5 minute(s) and 0 second(s)
Bands
Specific: no observed kDa Non-specific: 37, 50, 100 kDa
Positive control
CH12F3 Cells stimulated with CIT (Recombinant human TGFbeta1 (R&D systems #240-B) 1 ng/mL + Recombinant mouse IL-4 (Preprotech #214-14) 10 ng/mL + Functional Grade purified a-mouse CD40 (eBiosciences #16-0401) 1 µg/mL) for 16hs to induce AIID expression.
Additional data
Additional Notes
The Antibody was tested side by side with another commercial Ab against AID that is usually used for Western Blot. The positive sample are CH12 cells stimulated with CIT for 24hs where AID should have the highest protein expression. As negative controls were employed CH12 cells AID KO. Also, were included CH12F3 cells without stimulation to control background signal.
The Ab ab5197 does not show band at the expected size (24KDa) as is observed with the other commercial Ab used as reference. Instead, several non-specific bands are observed at higher molecular weights.
Abcam response
Thank you for participating in our AbTrial program and your valuable feedback. As the product had not yet been tested in this species we were unsure what the result might be. Although the homology was very high between human and mouse orthologs, unfortunately the results have not been satisfactory. We appreciate the time and effort that the reviewer has put in to testing this product in this application. We encourage all researchers to contact us should they wish to test any of our products in untested species or applications, as these may qualify for our AbTrial testing program.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Noé Seija
Verified customer
提交于 Jan 15 2021