Semmelweis University
Department of Medical Biochemistry
SOLID TUMORS OR HEALTHY TISSUE FOR RPPA

Ship tumors/tissues (min 20 mg, max 200 mg) in the designated cryovials following the instructions shown in 'sample submission' link, in dry ice (minimum 5 kg box). Please do not overfill the cryovial with tumor or tissue, see image below.
Protocols
CELL PELLET PREPARATION FOR RPPA

Attached cells:

1. Collect a minimum of 2 million cells by either Trypsin EDTA or scraping
2. Wash cell pellet twice with PBS (re-suspend and centrifuge, 2.5 min @3,500 rpm)
3. Aspirate PBS as much as possible without disturbing cell pellet
4. Store dry cell pellet in -80 oC
5. Ship samples in dry ice (minimum 5 kg box)

Suspension cells:

1. Collect a minimum of 2 million cells by centrifugation (2.5 min @3,500 rpm)
2. Wash cell pellet twice with PBS (re-suspend and centrifuge)
3. Aspirate PBS as much as possible without disturbing cell pellet
4. Store dry cell pellet in -80 oC
5. Ship samples in dry ice (minimum 5 kg box)

Along with the cell pellets please send us the information requested as detailed in 'sample submission' link  in this excel sheet (tab 'Other'). We prefer to obtain this information prior to receiving the samples.
Along with the tumor/tissues please send us the information requested as detailed in 'sample submission' link  in this excel sheet (tab 'Living human subject' or 'post-mortem', depending on the origin of the sample). We prefer to obtain this information prior to receiving the samples.
RPPA: Overview
Sample submission
Antibody information
Protocols
Technical information
Forms
Contact
For Participants
Main
Immunostaining spotted slides without Autostainer
The protocols appearing below are performed by our RPPA team:
Generation of lysates with the precellys 24 using solid samples
IMAGING AND QUANTIFICATION OF SPOTS USING INNOSCAN 710-IR
SPOTING OF LYSATES ON SLIDES USING THE 2470 AUSHON
IMMUNOSTAINING RPPA USING DAKO AUTOSTAINER
Antibody validation by Western Blot using GoBlot
Benzonase buffer estimator
Generation of lysates using pulverized samples
Generation of lysates using liquid samples (i.e. cell culture pellets)
Include only one tube per tumor or healthy tissue or stroma. Do not submit multiple tubes from the same tumor or healthy tissue or stroma. Also, please try to avoid including blood with the samples, but there is no need to make them completely blood-free.
Protein loading calculator for WBs
Gel running and transfer using iBlot
Staining spotted slides with FCF
Backup machine files (epMotion)_Lysates load to two plates_normalizations_buffer transfers_dilutions
Backup machine files (epMotion)_2_WP96 to 1_WP384
Backup machine files (epMotion)_BIOSIGMA
Loading of lysates to WP96 and WP384 using the epMotion 5073