User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/01

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Bradford, qPCR AKAP12 & AKAP9, Silverstaining
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Bradford

 * Bradford performed by Tina
 * 5,05 mg/mL protein in Lysis buffer
 * 4,02 mg/mL protein in HeLa -S sample

SDS-PAGE

 * 8% SDS-PAGE
 * preparation of cDNA and qPCR

Coomassie staining

 * As described 25June2010
 * For silver staining fix again in 40% Ethanol/10% acetic acid (2x 10-30 min) & 10% EtOH/5% acetic acid (2x 10 min)

Silver staining
1.	Fix gel for 2 h in fixing solution (30% ethanol (v/v) & 10% acetic acid (v/v)) 2.	Wash gel 2x 10 min. with 250 mL 10% 30 Ethanol (v/v) 3.	Wash gel 3x 10 min. with dH2O 10 min. OPTIONAL a.	Soak for 10 min in 3.4 mM potassium dichromate-0.0032 N nitric acid (200 ml/gel, 25°C) (stronger signal) b.	Wash with deionized water three times for 10 min (400 ml/gel, 25°C). STAINING 4.	Incubate gel 30 min. in 200 mL 0.1% Silvernitrate solution (w/v) 5.	Rinse 3x 5 min. with dH2O DEVELOPING 6.	Develop gel for 5 – 10 min. in 200 mL 3% NaCO3 + 200 μL 37% Formaldehyde solution 7.	Stop development with 200 mL 1~5% Acetic acid for 5~10 min. 8.	Wash gel with dH2O (2 min.) DESTAINING 9.	Destain with (Farmer’s reducer, will clear whole gel!) a.	50 mL 2% Potassium hexacyanoferrate(III) (C6N6FeK3) b.	50 mL 3% sodiumthiosulphate (Na2S2O3) 10.	Wash 3x 10 min. with dH2O (RESTAINING) (Drying the gel, gel can be soaked in drying solution (10% glycerol + 15% EtOH) for 30 min. and let to dry
 * FIXING (see above when following Coomassie staining)

Preparing cDNA

 * SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen)
 * Incl. SuperScript® III Reverse Transcriptase (Invitrogen) (DRAWER #5)

The following procedure is designed to convert 1 pg to 5 μg of total RNA or 1 pg to 500 ng of poly(A)+ RNA into first-strand cDNA:
 * Mix and briefly centrifuge each component before use.
 * Combine the following in a 0.2- or 0.5-ml tube:
 * Incubate at 65°C for 5 min (progr. ALI), and then immediately place on ice for at least 1 minute. Collect the contents of the tube by brief centrifugation.
 * Add the following to the tube on ice:
 * 2X First-Strand Reaction Mix 			10 μl
 * SuperScript™ III/RNaseOUT™ Enzyme Mix 	 2 μl
 * Vortex the sample briefly to mix, and collect by brief centrifugation (1 min. 1000rcf). Incubate as follows:
 * Random hexamer primed: 		5-10 min at 25°C, followed by 50 min at 50°C (Progr. VANN)
 * Collect the reactions by brief centrifugation
 * cDNA synthesis reaction can be stored at -20°C or used for PCR immediately

Real time qPCR

 * Thermo Scientific Solaris qPCR Gene Expression Assay
 * Two primers at 800 nM + one probe 200 nM = primer probe set (20x)
 * Solaris qPCR Gene Expression Master Mix (2x) (ROX)
 * qPCR cycler (ABI StepOne)
 * MicroAmp™ Fast optical 48-well reaction plate (0.1 mL) (Applied Biosystems)

Primer

 * Primer AKAP9 (Dharmacon Inc.
 * FW: AACCTGAAGATGTGCCTCCTG
 * REV: CTGGAGTGCATACCTTTC
 * Probe: GATTTTGTCTAATGAAA
 * AKAP12 (Dharmacon Inc.
 * FW CAAGCACAGGAGGAGTTACAG
 * REV CTGGTCTTCCAAACAGACAATG
 * Probe ATCATGCAGTTAAACTCA
 * 1) Thaw reagents on ice, mix (DO NOT VORTEX) and spin down
 * Mix
 * Solaris qPCR Master mix (2x)	12.5 μL
 * Solaris primer/probe set (20x)	1.25 μL
 * Water (PCR Grade)              9.25 μL
 * cDNA 2 μL
 * Total volume 25 μL
 * 1) Seal with optical clear seals and briefly centrifuge (removing bubbles)
 * 2) Run on thermal cycler (ABI StepOne)
 * 3) Data is collected during the last 40 cycles

Results

 * Coomassie staining

Discussion

 * Need a house hold gene to compare with

Related topics

 * Silver staining
 * http://www.ciwemb.edu/labs/koshland/protocols/protein/silverstain.html
 * http://www.crypto.wustl.edu/Protocols%20PDFs/Silver_staining_(SK).pdf
 * De Moreno et al.; Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie blue-silver stain procedure Anal Biochem. 1985 Dec;151(2):466-70 (PMID: 2420227)

Attachment
   [[Media:Bradford_01072010.xls| Bradford results, slightly different than those calculated by Tina]]

   [[Media:01072010_qPCR_AKAP12,_AKAP9.xls| qPCR results, without GAPDH]]


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