Anti-RYBP antibody [EPR13059(2)] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RYBP antibody [EPR13059(2)] - BSA and Azide free (AB250871)

Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling RYBP with purified ab185971 at 1/20 dilution (11.7μg/mL) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) (black). Unlabeled control - Unlabelled cells (blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185971).

• IP

Lab

Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - BSA and Azide free (AB250871)

This data was developed using ab185971, the same antibody clone in a different buffer formulation.

RYBP was immunoprecipitated from HEK-293 (human embryonic kidney epithelial cell) whole cell lysate 10 μg with ab185971 1/1000 dilution. Western blot was performed on the immunoprecipitate using ab185971 at 1/1000 dilution. Capture antibody, 1 : 30 dilution (2μg in 0.35mg lysates).

Rabbit monoclonal IgG (ab172730) Isotype Control was used instead of ab185971 in HEK-293 whole cell lysate (Lane 3).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (<a href='/en-us/products/primary-antibodies/rybp-antibody-epr130592-chip-grade-ab185971'>ab185971</a>) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate (Low Expression Sample) 10 μg

Lane 2:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 32 kDa

false

Exposure time: 15s

• ChIP

Unknown

ChIP - Anti-RYBP antibody [EPR13059(2)] - BSA and Azide free (AB250871)

Chromatin was prepared from HEK293 cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab185971 (red), and 20 μl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185971).

• IP

Lab

Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - BSA and Azide free (AB250871)

This data was developed using ab185971, the same antibody clone in a different buffer formulation.

RYBP was immunoprecipitated from NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 μg with ab185971 1/1000 dilution. Western blot was performed on the immunoprecipitate using ab185971 at 1/1000 dilution. Capture antibody, 1 : 30 dilution (2μg in 0.35mg lysates).

Rabbit monoclonal IgG (ab172730) Isotype Control was used instead of ab185971 in NIH/3T3 whole cell lysate (Lane 3).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (<a href='/en-us/products/primary-antibodies/rybp-antibody-epr130592-chip-grade-ab185971'>ab185971</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (Low Expression Sample) 10 μg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 32 kDa

false

Exposure time: 24s

• ChIP

Unknown

ChIP - Anti-RYBP antibody [EPR13059(2)] - BSA and Azide free (AB250871)

Chromatin was prepared from NIH/3T3 cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab185971 (red), and 20 μl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185971).