Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293,HELA and JURKAT)
Loading amount
10 µg
Specification
HEK293,HELA and JURKAT
Gel Running Conditions
Reduced Denaturing (4-20%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Other product details
Dilution
1/1500
Incubation time
1 hour(s) and 0 minute(s) · Diluent: TBST+5%Milk
Secondary antibody
Name
Non-Abcam antibody was used: Anti-Rabbit IgG
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/16000
Detection
Detection method
ECL Prime
Exposure
1 minute(s) and 0 second(s)
Bands
Specific: No kDa Non-specific: 75, 70, 30 kDa
Positive control
1-HEK293 Lysate
2-HELA Lysate
3-JURKAT Lysate
2-HELA Lysate
3-JURKAT Lysate
Additional data
Additional Notes
Unsure as to the identity of these non-specific bands, but they are possibly cleaved fragments.
Abcam response
Thank you for your valuable feedback.
Since the protein target has such a high molecular weight (predicted around 278 kDa), it may be necessary to alter the protocol to ensure that the protein transfers to the membrane. You can add 0.1% SDS to the transfer buffer and decrease the methanol percentage to 10% or less to improve transfer.
Decreasing the antibody dilution to 1:500 and loading 20-30 ug of protein will ensure that the 278 kDa protein can be detected after transfer.
We do guarantee this antibody to work in Western blot with human samples according to our Abpromise, so if these suggestions do not improve the results we would be happy to send a replacement or issue a credit or refund.
Since the protein target has such a high molecular weight (predicted around 278 kDa), it may be necessary to alter the protocol to ensure that the protein transfers to the membrane. You can add 0.1% SDS to the transfer buffer and decrease the methanol percentage to 10% or less to improve transfer.
Decreasing the antibody dilution to 1:500 and loading 20-30 ug of protein will ensure that the 278 kDa protein can be detected after transfer.
We do guarantee this antibody to work in Western blot with human samples according to our Abpromise, so if these suggestions do not improve the results we would be happy to send a replacement or issue a credit or 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.
Abcam user community
Verified customer
提交于 Sep 07 2011