Application
Western blot
Sample
Zebrafish Tissue lysate - whole (brain)
Loading amount
21 µg
Specification
brain
Gel Running Conditions
Reduced Denaturing (10% resolving SDS-PAGE gel)
Blocking step
Milk as blocking agent for 48 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Other product details
Dilution
1/50000000
Incubation time
17 hour(s) and 15 minute(s) · Temperature: 4°C · Diluent: 5%milk powder inTBS-T
Secondary antibody
Name
Non-Abcam antibody was used: Anti-rabbit IgG, Cell Signaling 7074
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/2500
Detection
Detection method
West Femto
Exposure
45 second(s)
Bands
Specific: 38 kDa
Additional data
Additional Notes
PVDF membrane was used. Tissue lysates were prepared both with RIPA buffer and Camiolo buffer, both of them worked. First two lanes in the picture were loaded with Zebrafish brain lysate obtained with RIPA buffer, lanes 3 and 4 were loaded with Zebrafish brain lysate obtained with Camiolo buffer. Molecular weight marker is page ruler prestained protein ladder (10-170 kDa, Fermentas).
Primary antibody dilution corresponds to 1/50000000. Actually, we have reached this ratio by doing more than one experiment. Firstly, we tried the dilution ratio (1/5000) recommended on the product sheet. We observed many bands and background. Based on troubleshooting recommendations from Abcam, we diluted it 100 folds further. We observed two bands this time, one at 35 kDa and one below it. We decided to dilute it 100 folds further in order to get rid of the extra band and it gave the result that I sent to you. So, resulting dilution is 1/50000000.
First lane in the gel is page ruler prestained protein ladder (10-170 kDa, Fermentas). Second and third lanes in the picture were loaded with Zebrafish brain lysate obtained with RIPA buffer (20.54 ug each), lanes 4 and 5 were loaded with Zebrafish brain lysate obtained with Camiolo buffer (21.12 ug each). Our test code for this antibody is 40801.
Primary antibody dilution corresponds to 1/50000000. Actually, we have reached this ratio by doing more than one experiment. Firstly, we tried the dilution ratio (1/5000) recommended on the product sheet. We observed many bands and background. Based on troubleshooting recommendations from Abcam, we diluted it 100 folds further. We observed two bands this time, one at 35 kDa and one below it. We decided to dilute it 100 folds further in order to get rid of the extra band and it gave the result that I sent to you. So, resulting dilution is 1/50000000.
First lane in the gel is page ruler prestained protein ladder (10-170 kDa, Fermentas). Second and third lanes in the picture were loaded with Zebrafish brain lysate obtained with RIPA buffer (20.54 ug each), lanes 4 and 5 were loaded with Zebrafish brain lysate obtained with Camiolo buffer (21.12 ug each). Our test code for this antibody is 40801.
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.
Mrs. Fusun Doldur Balli
Verified customer
投稿 Apr 12 2012