summary of print runs 9-2-13

2014-08-29

++ summary of print runs 9-2-13


summary of print runs 


Print Run 1 Aminosilane slides


comment: smearing and intensities lower than desired


-there would have been two much lysozyme in this print run





Print Run 2


-too much lysozyme in this print run





Print run 3


-potential title: CodeLink and Aminosilane test


-experiment on 7-8-11


-CodeLink slides used and aminosilane slides tested


-blocked 30 min with CodeLink ethanolamine blocking buffer (0.1 M Tris, 50 mM EtOH Amine Methol" (ethanolamine?), pH 9, blocked 30 min with BSA containing buffer


-4 slides 5 nM primary (purified rabbit anti-h-SMC1fs 17 mer)


-6 slides 1:500 sera (rabbit anti-h-SMC1fs 27mer non-purified)


-5 nM secondary (Bethyl biotinylated goat anti-Rabbit IgG)


-5 nM tertiary (streptavidin AF647)








Cell lysate print 4


-potential title: Denatured lysate


-denatured and non-denatured proteins were printed onto the slides.


-This time denatured and non-denatured proteins were printed onto the slides.


-lysate denatured with SDS (final concentration of 1% SDS and heated at 50 C 1hr)


-Basically, there was still a lot of smearing, and I was not able to detect a 1/10 dilution of SMC1fs into PUC19 lysate above background. 








Cell lysate print 5


-potential title: HEPES and glycerol buffer


-HEPES and glycerol were tested in order to try to decrease smearing, but this did not seem to work at all


-2 Aminosilane: denatured HEPES, non-denatured HEPES, denatured glycerol, non-denatured glycerol


-2 CodeLink: denatured HEPES, non-denatured HEPES, denatured glycerol, non-denatured glycerol


-The HEPES and glycerol buffers did not seem to help my slides print well at all.  Some of the HEPES protein (GST, GST-SMC1fs, LL) CodeLink spots actually looked quite good.  However, most of the lysate was extremely smeared.  John suggested that the problem may not lie in the buffers alone.  He said that the high humidity that we've had over the last few days may have adversely affected the print run.


-primary antibody: 1:500 rabbit anti-hSMC1fs 17mer


secondary 5 nM biotin goat anti-Rabbit


tertiary 5 nM streptavidin AF647


-CodeLink blocking buffer (codelink buffer 30m then incubation 30m)


-aminosilane (30m blocking buffer then 30m blocking buffer)


-blocks printed on each array


--Hepes non-denature  	glycerol non-denatured


hepes denatured 	glycerol denatured


-makeup of one block


--1_D Non-de he	0.5_D Non-de he


0.1_D Non-de he	0.01_D Non-de he


0.001_D Non-de he	0.001_D Non-de he


0_D Non-de he	Blank_D Non-de he


GST_SMC1fs_D Non-de he	GST_D Non-de he


LL_1-200 Non-de he	Blank


-HEPES = 7 mM HEPES, 1 mM EDTA (pH adjusted to 7.4 with KOH)


-Glycerol = 5% Glycerol








Cell Lysate Print 6


-potential tilte: PEI polymer slides


-printed onto Valiery's polymer slides


-SMC1fs dilution series


Samples (9): 1X	0.5X	0.1X	0.01X	0.001X	0.0001X	0X	GST	GST-SMC1fs


-used denatured and non-denatured cell lysate (lysate denatured with SDS (final concentration of 1% SDS and heated at 50 C 1hr))


-block order: non-denatured, denatured 


-primary antibody: rabbit anti-hSMC1fs 17mer


-secondary goat anti-rabbit IgG 5nM


-tertiary: streptavidin AF647 5 nM


-blocking buffer ((Valiery's slides 30m blocking buffer, 30m blocking buffer), (CodeLink HD Array Blocking Buffer 30m, Incubation buffer 30m)








Cell lysate print 7


-potential title: nitrocellulose test


-experiment on 9-23-11


-The nitrocellulose slides worked really well with good morphology and little smearing. However, for some reason only the positive control exhibited signal. The SMC1fs dilution series did not show any signal. Perhaps this could be because this protein was produced from bacteria transformed with a plasmid from colonies that had been on a plate for several months.


-denatured and non-denatured proteins printed


--dilution series: 1X


	0.5X


	0.1X


	0.01X


	0.001X


	0.0001X


	0


	GST


	GST-SMC1fs


	Old 1X from 8-10-11


-2 nitrocellulose (1 post print wash in Matt's buffer and 1 no post print wash)


-2 CodeLink (no post print wash


-2 Valiery (post print wash)


-CodeLink slides washed in CodeLink blocking 30m and blocking buffer 30m; non-codelink slides washed in blocking 30m and blocking 30m


-primary: 1:500 sera


-secondary: 5 nM


-tertiary: 5 nM








Cell Lysate Print 8


-potential title: fresh SMC1fs lysate on nitrocellulose slides


-experiment on 10-15-11


-fairly fresh SMC1fs lysate diluted in plain non-induced pET32b lysate printed onto nitrocellulose; intensity was detected well and morphology looked good


-The intensity of the 1X and 0.5X spots are also clearly high enough to detect over a negative spot.


-bar graph of detected signals here:


"F:\kurt\storage\CIM Research Folder\DR\2013\4-27-13\wiki_download\cell lysate printing 8\Block 1 data.xlsx"


"F:\kurt\storage\CIM Research Folder\DR\2013\4-27-13\wiki_download\cell lysate printing 8\Block 2 data.xlsx"


"F:\kurt\storage\CIM Research Folder\DR\2013\4-27-13\wiki_download\cell lysate printing 8\Blocks 1 and 2.xlsx"


-slide washed with post print wash buffer


-primary 1:500 purified rabbit h-SMC1fs


-secondary 5 nM anti-rabbit


-tertiary 5 nM AF647








Cell Lysate Print 9


-potential title: Nitrocellulose with 800 um spacing


-experiment on 11-3-11


-spots were spaced farther apart (800 um) on nitrocellulose slides; this distance was great enough to keep the spots apart


-graphs


--"F:\kurt\storage\CIM Research Folder\DR\2013\5-26-13\wiki_download\block_1_11-3-11.PNG"


--"F:\kurt\storage\CIM Research Folder\DR\2013\5-26-13\wiki_download\block_2_11-3-11.PNG"











Cell lysate print 10


title: Concentrated primary with nitrocellulose


-experiment around 11-6-11


-In this run, I increased the concentration of the primary antibody from 1:500 to 1:20. I also increased the incubation time of the primary from 1 hr to 2hr. I was able to detect much lower dilutions of protein lysate than I ever have before. The slide did not have an even intensity though. I think the primary concentration may have been to high. Perhaps I could even do a 1:500 dilution of primary if I did an overnight incubation. In the future, I may want to try a range of primary concentrations and times.


-800 um spacing


-primary ab = Balb Nat anti-mSMC1 27mer low anti-Thap2+Rvm4 pool of 52-6, 53-3, and 54-5


-secondary = goat anti-mouse bethyl (1:100)


-tertiary = AF647 5 nM


-graphs here:


http://azim58.wikispaces.com/Cell+lysate+printing+10








Cell Lysate Print 11


title: Nitrocellulose with overnight primary


-12-13-11 of notebook


-Grace BioLabs Nitrocellulose Slides with overnight primary 1:500 dilution with low agitation


-The 1/100 dilution was detectable above lysate spots with no SMC1fs protein. However, the 1/1000 dilution was not detectable. I will try another experiment with a more concentrated primary. I will also test out the Super G Blocking buffer.


-primary: 1:500 mouse Balb Nat anti-SMC1fs 5 nM


-secondary: goat anti-mouse AF647


-graphs


--"S:\Research\Cancer_Eradication\Users\kwhittem\kwhittem\Raw Data Often Originally on Research Drive\2011\12-14-11\Tecan Power Scanner\SMC1fs Cell Lysate on Nitrocellulose slide scanned 10 laser power block 1.xls"


--"S:\Research\Cancer_Eradication\Users\kwhittem\kwhittem\Raw Data Often Originally on Research Drive\2011\12-14-11\Tecan Power Scanner\SMC1fs Cell Lysate on Nitrocellulose slide scanned 10 laser power block 2.xls"








Cell lysate print 12


title: Nitrocellulose with Super G Blocking Buffer and 2hr incubation


-experiment on 12-19-11


-Grace BioLabs Nitrocellulose Slides with Super G Blocking Buffer 2 hr primary at 37C Summary


-The Super G Blocking Buffer appears to decrease all intensities considerably. However, the ability to distinguish between different lysate dilutions and the ability to detect lower dilutions seems to increase. The error bars of replicate lysate spots also seems quite small.


-primary sera: BALB Nat anti-mSMC1 27 mer Low anti-t Hap2 + Rbrwh Pooled 10/15/11


-secondary: goat anti-mouse AF647


-top block: 1:20 dilution; bottom block 1:500 dilution


-graphs:


--http://azim58.wikispaces.com/file/view/Top_Block_Graph.png/406760300/Top_Block_Graph.png


--F:\kurt\storage\CIM Research Folder\DR\2013\5-27-13\wiki_download\Bottome Block Graph 2 12-18-11.png








Cell lysate print 13


-(Grace BioLabs Nitrocellulose Slides 16 hr room tem1perature incubation; one with Super G blocking buffer one without)


-Slides ran around 1-20-12


-500 um spacing


-Primary: Balb/Nat SMC1 27 mer


-Secondary: goat anti mouse


-I was going to align and analyze these arrays, but after looking at the images of the slides, I see that the spots were very faint and quite smeared. Did the overnight condition cause these undesirable results? Johnny and I will print 4 more slides for a test.








Cell lysate print 14


title: Nitrocellulose blocking buffer and incubation time test


-experiment on 2-11-12


-Grace BioLabs Nitrocellulose Slides; blocking tested with Super G and BSA; primary incubation time tested with 1 hr 37 and 16 hr 23 C


-Slides


1 slide BSA primary incubation 1 hr


1 slide Super G primary incubation 1 hr


1 slide BSA primary incubation 16 hr


1 slide Super G primary incubation 16 hr


-nitrocellulose slides just washed with H20


-primary: 1:500 sera


-basically The Super G results in less smearing, and the overnight conditions resulted in higher signals. I was able to detect 1/1000 and even 1/10000 SMC1fs dilutions above background.


-The ratio of the signal to the background increased when going from 1 hr to overnight incubations with both the BSA and the Super G.


The signal to noise ratio of the BSA samples was higher than the signal to noise ratio of the Super G samples.  However, this makes sense since the Super G decreases overall signal intensity resulting in a signal that will be closer to the background signal.  However, in my experience, the super g does allow for the detection of proteins with a lower concentration.  The Super G blocking buffer also generally results in less smearing.


     Based on these results and general knowledge from previous experiments, I will perform the actual library hybridizations overnight with Super G blocking buffer.


-graphs found here


F:\kurt\storage\CIM Research Folder\DR\2012\8-25-12\8-25-12_1140\Printing Library onto array\Cell Lysate Test Prints\blocking and time test 2-11-12



Inkscape document containing images from these print runs