Western blot abreview for Anti-PSMD14 antibody [EPR4257]

Excellent
Abreviews
Application
Western blot
Sample
Saccharomyces cerevisiae Purified protein (protein purified from yeast or recombinantly expre)
Gel Running Conditions
Reduced Denaturing (4-20% Tris Glycine Biorad Stain free)
Loading amount
0.4 µg
Specification
protein purified from yeast or recombinantly expre
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 6% · Temperature: 4°C

Other product details

Incubation time
2 hour(s) and 20 minute(s) · Temperature: 24°C · Diluent: TBST
Dilution
1/5000

Secondary antibody

Dilution
1/10000
Name
Non-Abcam antibody was used: ECLtm Anti-Rabbit IgG, HRP-linked whole antibody (
Host species: Equus
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase

Detection

Detection method
ECL+
Negative control
2
Exposure
40 second(s)
Bands
Specific: 27, 34, 36 kDa
Positive control
1 (lysate sample failed) 4-11(purified protein samples worked)

Additional data

Additional Notes
Purified and lysate samples are loaded and should contain S. cerevisiae Rpn11(homolog of PSMD14). Lysate samples showed no signal, but all purified proteins appear at the correct molecular weight (MW). non-specific bands were not seen in any samples. After development with the ab109123 (top), blot was stripped reblotted against M2-FLAG (bottom). For lanes 4 and 5, FLAG signal was not present, as expected, but bands of the same MW as the antiPSMD14 samples were present for lanes 6-11. No signal was observed in lanes 1 and 2, which was unexpected for lane 1. Lower MW species seen for lane 6, and a ~100kDa band seen in lane 6 were also present in the anti FLAG blot, therefore it is likely that those bands are truncated or modified Rpn11 not non-specific bands. Lanes: 1: 3uL of S. cerevisiae lysate expressing Rpn11 (H109A, H111A)-FLAG 2: 3uL of the above lysate after FLAG IP 3: ladder (not visible in Chemiluminescence) 4: Rpn11 residues 1-239 untagged, Recombinantly expressed in E. coli, purified protein that forms a dimer with Rpn8. MW: 27kDa 5: Rpn11, untagged, Recombinantly expressed in E. coli, purified protein as proteasome 'lid' complex MW: 34kDa 6: Rpn11, with C-terminal FLAG-HIS tag, endogenous purified protein, purified as proteasome 'lid' MW: 38kDa, sample loaded with 3x the protein of other lanes 7: Rpn11 (H109A, H111A), with C-terminal FLAG-HIS tag, endogenous purified as proteasome 'lid' complex. MW: 36kDa. 8, 9: Rpn11 (H109A, H111A), with C-terminal FLAG-HIS tag, endogenous purified protein as 26S proteasome holoenzyme complex MW: 36kDa. 10, 11: Rpn11 (H109A, H111A), with C-terminal FLAG-HIS tag,endogenous purified protein as proteasome 'lid' complex purified protein and independent prep from #8 & #9 protein. MW: 36kDa. ~1/3 as much protein as other samples (excluding #6)

Ellen Goodall

Verified customer

Submitted Aug 01 2017

Sign up