Antibody-Based Proteomics

Sample Preparation

Service Procedure includes (buffer is not included):

1. Please prepare your tissue samples in the tubes specified in the protocol. For BCM investigators, if you don't have the type of tubes, we maybe able to provide TissueLyzer Tube and Labels.

2. TissueLyzer Homogenization

3.Tissue Lysis Preparation

4. BCA Protein Concentration Measurement

5. Calculate protein concentration and dilution

6. Prepare lysates into 0.5mg/ml with RPPA lysis buffer.

7. Store the remaining lysates at a carbboard box and store them at -80 C

1. Download the Antibody Inventory Form. Please be advised that recommended WB dilutions are determined by signals received from prior RPPA scans. Please optimize your dilutions if they do not work properly.
2. Fill out Table with needed Antibodies. Blank Table can be downloaded to be filled and uploaded.
3. Click SUBMIT REQUEST TO COREat the very bottom of this page. YOUR REQUEST WILL NOT BE RECEIVED UNTIL YOU CLICK 

Note:

• Not all antibodies available upon request
• Our regular turnaround time for antibody request is 6-10 business days (i.e. order on a Monday and available on next Tuesday-Friday) or longer if we are in the middle of a RPPA run. Core staff will inform you as soon as we have the aliquot(s) available to pick up. 

This panel includes histone marks which require purified histone to generate reliable data.

The service procedure for cells and tissues includes (buffer cost not included):

1. Washing cell pellet or homogenizing tissue sample.
2. Nuclear extraction using NIB-250 buffer.
3. Acid extraction of histones. 
4. Histone quantitation (see charge).
5. Preparing histones with RPPA sample buffer and preparing 2 tubes per sample as ready-to-use RPPA samples.
6. Storing remaining histones in a cardboard box at -80 C.
7. Returning ready-to-use histone samples after completion of project. 

The histone purification buffer contains the following:

NIB-250

• 15mM Tris-HCl (pH 7.5)
• 60mM KCl
• 15mM NaCl
• 1mM CaCl₂
• 5mM MgCl₂
• 250mM Sucrose

Inhibitors

• Protease inhibitor
• Phosphatase inhibitor
• Sodium Butyrate (HDAC inhibitor)

This service includes running histone extracts on a gradient SDS-PAGE gel for 8 samples followed by Coomassie blue staining. Bands will then be quantitated using image analysis software. 

Includes one of each of the following:

• RPPA Lysis Buffer (3ml)
• 5x Protease Inhibitor (1ml)
• 5x Phosphatase Inhibitor (1ml)
• 2x SDS Sample Buffer (1ml)
• 4x SDS Sample Buffer with Bromophenol Blue (0.5ml)

Total proteins and phosphoproteins in the various (not limited to) protein pathways or functional protein groups: epithelial-mesenchymal transition (EMT), stem cells, apoptosis, DNA damage, autophagy, proliferation and cell cycle, growth factor receptors, cytokines/STATs and nuclear receptors/transcriptional and chromatin regulatory proteins, and histone modifier proteins (epigenetic panel). 

This panel does not include histone marks which require purified histone to generate reliable data.

Testing samples to determine sample quality prior to proceeding with RPPA analysis.

Please work with core director to determine whether a test printing is necessary. 

Most standard samples (from cell lines and tissue samples) don't need test printing, if our protocol is followed. Other sample types may need to be tested.  

The service include (up to 20 samples):

1. Preparing RPPA sample map

2. Sample centrifugation and denaturing for RPPA

3. Transferring samples to 384 well plates for printing 

4. Setup instrument and generate .gal file.

5. Printing samples on 2 slides

6. Performing total protein staining for the two slides

7. Scanning slides to generate images

8. Performing image analyses for the images

9. Generating analyzable data format for quality assessment

10. Calculate cv% of the technical triplicate to determine sample quality. 

Service Procedure includes (buffer is not included):

1. Please prepare your tissue samples in the tubes specified in the protocol. For BCM investigators, if you don't have the type of tubes, we maybe able to provide TissueLyzer Tube and Labels.

2. TissueLyzer Homogenization

3.Tissue Lysis Preparation

4. BCA Protein Concentration Measurement

5. Calculate protein concentration and dilution

6. Prepare lysates into RPPA sample buffer and prepare 2 tubes per sample at RPPA-ready status.

7. Store the remaining lysates at a carbboard box and store them at -80 C

8. Ready to return the remaining samples to investigator. 

Sample Preparation

Service Procedure includes (buffer is not included):

1. Please prepare your tissue samples in the tubes specified in the protocol. For BCM investigators, if you don't have the type of tubes, we maybe able to provide TissueLyzer Tube and Labels.

2. TissueLyzer Homogenization

3.Tissue Lysis Preparation

4. BCA Protein Concentration Measurement

5. Calculate protein concentration and dilution

6. Prepare lysates into 0.5mg/ml with RPPA lysis buffer.

7. Store the remaining lysates at a carbboard box and store them at -80 C

1. Download the Antibody Inventory Form. Please be advised that recommended WB dilutions are determined by signals received from prior RPPA scans. Please optimize your dilutions if they do not work properly.
2. Fill out Table with needed Antibodies. Blank Table can be downloaded to be filled and uploaded.
3. Click SUBMIT REQUEST TO COREat the very bottom of this page. YOUR REQUEST WILL NOT BE RECEIVED UNTIL YOU CLICK 

Note:

• Not all antibodies available upon request
• Our regular turnaround time for antibody request is 6-10 business days (i.e. order on a Monday and available on next Tuesday-Friday) or longer if we are in the middle of a RPPA run. Core staff will inform you as soon as we have the aliquot(s) available to pick up. 

This panel includes histone marks which require purified histone to generate reliable data.

The service procedure for cells and tissues includes (buffer cost not included):

1. Washing cell pellet or homogenizing tissue sample.
2. Nuclear extraction using NIB-250 buffer.
3. Acid extraction of histones. 
4. Histone quantitation (see charge).
5. Preparing histones with RPPA sample buffer and preparing 2 tubes per sample as ready-to-use RPPA samples.
6. Storing remaining histones in a cardboard box at -80 C.
7. Returning ready-to-use histone samples after completion of project. 

The histone purification buffer contains the following:

NIB-250

• 15mM Tris-HCl (pH 7.5)
• 60mM KCl
• 15mM NaCl
• 1mM CaCl₂
• 5mM MgCl₂
• 250mM Sucrose

Inhibitors

• Protease inhibitor
• Phosphatase inhibitor
• Sodium Butyrate (HDAC inhibitor)

This service includes running histone extracts on a gradient SDS-PAGE gel for 8 samples followed by Coomassie blue staining. Bands will then be quantitated using image analysis software. 

Includes one of each of the following:

• RPPA Lysis Buffer (3ml)
• 5x Protease Inhibitor (1ml)
• 5x Phosphatase Inhibitor (1ml)
• 2x SDS Sample Buffer (1ml)
• 4x SDS Sample Buffer with Bromophenol Blue (0.5ml)

Total proteins and phosphoproteins in the various (not limited to) protein pathways or functional protein groups: epithelial-mesenchymal transition (EMT), stem cells, apoptosis, DNA damage, autophagy, proliferation and cell cycle, growth factor receptors, cytokines/STATs and nuclear receptors/transcriptional and chromatin regulatory proteins, and histone modifier proteins (epigenetic panel). 

This panel does not include histone marks which require purified histone to generate reliable data.

Testing samples to determine sample quality prior to proceeding with RPPA analysis.

Please work with core director to determine whether a test printing is necessary. 

Most standard samples (from cell lines and tissue samples) don't need test printing, if our protocol is followed. Other sample types may need to be tested.  

The service include (up to 20 samples):

1. Preparing RPPA sample map

2. Sample centrifugation and denaturing for RPPA

3. Transferring samples to 384 well plates for printing 

4. Setup instrument and generate .gal file.

5. Printing samples on 2 slides

6. Performing total protein staining for the two slides

7. Scanning slides to generate images

8. Performing image analyses for the images

9. Generating analyzable data format for quality assessment

10. Calculate cv% of the technical triplicate to determine sample quality. 

Service Procedure includes (buffer is not included):

1. Please prepare your tissue samples in the tubes specified in the protocol. For BCM investigators, if you don't have the type of tubes, we maybe able to provide TissueLyzer Tube and Labels.

2. TissueLyzer Homogenization

3.Tissue Lysis Preparation

4. BCA Protein Concentration Measurement

5. Calculate protein concentration and dilution

6. Prepare lysates into RPPA sample buffer and prepare 2 tubes per sample at RPPA-ready status.

7. Store the remaining lysates at a carbboard box and store them at -80 C

8. Ready to return the remaining samples to investigator.