Application
Western blot
Sample
Human Cell lysate - whole cell (HELA)
Loading amount
50 µg
Specification
HELA
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 23°C
Other product details
Dilution
1/100
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBST
Secondary antibody
Name
Non-Abcam antibody was used: GOAT ANTI-RABBIT
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/2000
Detection
Detection method
ECL
Exposure
1 minute(s) and 0 second(s)
Bands
Specific: 86? kDa Non-specific: 64 kDa
Additional data
Additional Notes
A 1:1000 dilution of primary antibody gave a strong 64KD band, which the antibody also pulled during IP. Since the reported 100KD band was not detected, the membranes were incubated with a 1:100 dilution of primary to reveal a 86 KD band (which may be the receptor), along with several other nonspecific bands. It may be an issue with just this aliquot of antibody, as even ICC with this antibody resulted in nonspecific staining.
Abcam response
As you have been seeing a band in the expected region at a dilution of 1/100 I would continue to try with this dilution and try to reduce the amount of background observed. This may be improved by trying BSA (3% in TBS-T) as the blocking agent, incubating for 1 hour at room temperature as you have been doing.
I would also suggest diluting your antibody in 1% BSA TBS-T and try incubating at room temperature for 1 hour as well as 4°C overnight as you have been doing.
If the antibody continues to produce poor results please do not hesitate to let me know. If the antibody was purchased within the last 6 months and has been used as recommended on the datasheet you would be eligible for a replacement antibody (this one or an alternative from our catalogue) or a refund.
I would also suggest diluting your antibody in 1% BSA TBS-T and try incubating at room temperature for 1 hour as well as 4°C overnight as you have been doing.
If the antibody continues to produce poor results please do not hesitate to let me know. If the antibody was purchased within the last 6 months and has been used as recommended on the datasheet you would be eligible for a replacement antibody (this one or an alternative from our catalogue) or a refund.
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.
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投稿 Jan 16 2012