|
| Status |
Public on Dec 31, 2010 |
| Title |
EREB2-5 cells transfected with Rta vector, minus estradiol |
| Sample type |
protein |
| |
|
| Source name |
EREB2-5 cells
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: EREB2-5 cells grown in the absence of beta-estradiol
cell line: EREB2-5
transfection: Rta vector
|
| Extracted molecule |
protein |
| Extraction protocol |
cell lysates prepared as suggested by the array manufacturer
|
| Label |
bioin-labeled anti-apoptosis markers
|
| Label protocol |
bioin-labeled anti-apoptosis markers from RayBio Human Apoptosis Antibody Array Kit
|
| |
|
| Hybridization protocol |
A. Blocking and Incubation
1. Place each membrane into the provided 8-well tray (top left corner marked with “-”).
Note: The printed side should be facing upward. 2. Add 1 ml Blocking Buffer and incubate at room temperature with
gentle shaking for 30 minutes to block membranes.
3. Decant Blocking Buffer from each well. Add 1 ml diluted sample onto each array membrane, and cover with the lid. Incubate at 2 – 8 0C overnight on a rocker or shaker (low speed 1-2 cycles/sec). Dilute sample using Blocking Buffer.
RayBio® Human Apoptosis Antibody Array Kit
Note: 1). Dilute cell lysates at least 5 folds with Blocking Buffer to avoid high background.
Note: 2). Optimal sample dilution factors should be determined empirically. More of the sample can be used if signals are too weak. If signals are too strong, the sample can be diluted further.
Note: 3). Incubation may be done at room temperature for 4 hours, but at the cost of impaired sensitivity of the signals.
Decant the samples from each well, and wash 3 times with 1.5 ml of 1X Wash Buffer I at room temperature with shaking, 5 min per wash.
Wash 2 times with 1.5 ml of 1X Wash Buffer II at room temperature with shaking, 5 min per wash.
Carefully remove wash buffer from each well containing array membranes.
Add 1 ml of diluted Cocktail of Biotin-Conjugated Antibody Mix to each membrane. Incubate at 2 – 8 0C with gentle shaking overnight.
Note: Incubation may be done at room temperature for 2 hours. Wash as directed in steps 5 and 6. Add 1.5 ml of 1X HRP-conjugated Streptavidin to each membrane.
Note: Mix tube containing 1X HRP-Conjugated Streptavidin well before use since precipitation may form during storage.
10. Incubate at room temperature for 1.5 hours.
RayBio® Human Apoptosis Antibody Array Kit
Note: incubation may be done overnight at 40C.
12. Wash as directed in steps 5 and 6.
|
| Scan protocol |
B. Detection
* Do not let the membrane dry out during detection. The detection process must be completed within 40 minutes without stopping.
1. Proceed with detection reaction.
Add 250 ?l of Detection Buffer C and 250 ?l of Detection Buffer D for one membrane; mix both solutions; Drain off excess wash buffer by holding the membrane vertically with forceps. Place membrane protein side up (“-” mark is on the protein side top left corner) on a clean plastic sheet (provided in the kit). Pipette the mixed Detection Buffer on to the membrane and incubate at room temperature with gentle shaking for 2 minutes. Ensure that the detection mixture is completely and evenly covering the membrane without any air bubbles.
2. Drain off excess detection reagent by holding the membrane vertically with forceps and touching the edge against a tissue. Gently place the membrane, protein side up, on a piece of plastic sheet (“-” mark is on the protein side top left corner). Cover the array with another piece of plastic sheet. Gently smooth out any air bubbles. Avoid using pressure on the membrane.
3. Detect signal directly from membrane using chemiluminescence imaging system or expose to x-ray film (we recommend to use Kodak X-OmatTM AR film) detect signal using film developer. Expose the membranes for 40 Seconds. Then re-expose the film according to the intensity of signals. If the signals are too strong (background too high), reduce exposure time (e.g., 5–30 seconds). If the signals are too weak, increase exposure time
RayBio® Human Apoptosis Antibody Array Kit
(eg, 2–20 min or overnight). Or re-incubate membranes overnight with 1X HRP-conjugated Streptavidin, and repeat detection on the second day.
4. Save membranes at –20 °C to –80 °C for future reference.
|
| Description |
EREB2-5 cells were transfected and grown in the presence or absence of β-estradiol, as described. Seven days post-transfection, protein extracts were prepared, and 200 ugs. of each were analyzed using the RayBio Human Apoptosis Antibody Array Kit (RayBiotech) as per manufacturers suggestions. The membranes were exposed to autoradiography film for different times to detect the chemiluminescent signals. Images with signals in linear range were quantitated using the program ImageJ [59].
|
| Data processing |
For each membrane, signals from the negative control spots were averaged, and then subtracted from each of the other spots. A signal was considered valid if its value exceeded both its average local background, and the average of all valid negative control values. Valid signals were normalized using the positive control spots (for cellular BID protein). Fold change in signals for each spot were quantitated by dividing by the valid signals for each corresponding spot on the minus β-estradiol membrane. Average fold change, and standard deviation, were calculated for each protein.
|
| |
|
| Submission date |
Apr 17, 2010 |
| Last update date |
Dec 31, 2010 |
| Contact name |
David M. Lukac |
| E-mail(s) |
lukacdm@njms.rutgers.edu
|
| Phone |
973 972-4868
|
| Organization name |
Rutgers Univ./NJ Medical School
|
| Department |
Microbiology, Biochemistry and Molecular Genetics
|
| Street address |
225 Warren St.; ICPH E350C; PO Box 1709
|
| City |
Newark |
| State/province |
NJ |
| ZIP/Postal code |
07101 |
| Country |
USA |
| |
|
| Platform ID |
GPL10337 |
| Series (1) |
| GSE21375 |
Convergence of KSHV Reactivation with EBV latency and cellular growth mediated by the Notch signaling pathway in co-infected cells. |
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