| Date | Laborator | Experiment short name | Experiment details |
Tissue |
Gel type | Antibodies (pri/sec dilution) | Results (specific signal, background) | Problems | Conclusions |
| 020625 | MAL/EED | Establishing standard protocol-Extractions | Trizol and lysis buffer (Na-Acetate + Triton) protein extractions and BSA standards. | NaAc/Mu | - | - | BSA standards plot | - | - |
| 020729 | MAL/EED | Establishing standard protocol-Extractions | Trizol extraction | NaAc/Mu | - | - | No pellet! | Phases separated incorrectly, red phase up and aqeus phase down | - |
| 020730 | MAL/EED | Establishing standard protocol-Extractions | Detergent lysis using Na-Acetate buffer | NaAc/Mu | - | - | Abundant protein extracted without problems. Aliquots prepared and stored at -70ºC | - | Worked correctly, gave good yield: 9,9mg protein from 148mg tissue (0,067mg protein/mg tissue) |
| 020801 | MAL/EED | Testing anti-GFAP | Establishing standard protocol, on 25, 50 and 100µg protein from 020730. | NaAc/Mu | 8% Tris-Glycine | GFAP mo: 1:15000. HRP-conj-anti-Mo 1:1500. | Band of correct size identified using 25, 50 and 100µg protein. Low background. Weak unspecific bands only at 100µg. Film | Marker is unreliable -too weak on gel, weaker on membrane: too old? | Very promising results. Nice signal to noise ratio. Na-Acetate lysis buffer extraction works well. |
| 020805 | MAL/EED | Testing anti-Shc (pan) | Establishing standard protocol, on 25, 50 and 100µg protein from 020730. | NaAc/Mu | 8% Tris-Glycine | Shc-mo: 1:1000 (upstate #06-203). HRP-conj-anti-Mo 1:1500. | OK. Film | Marker is unreliable -too weak on gel, weaker on membrane: too old? | - |
| 020807 | MAL/EED | Testing anti-p75 | Establishing standard protocol, on 25, 50 and 100µg protein from 020730. | NaAc/Mu | 8% Tris-Glycine | p75-rb 1:10000 (gift Wesskamp)
HRP-conj-anti-Rb 1:1500 |
Band visible but size cannot be judged. | Marker is unreliable -too weak on gel, weaker on membrane: too old? | New marker needed! |
| 020808 | MAL/EED | SC extraction & testing of anti-Shc (pan) | Extraction: rem SC 20/65mg in Na-Acetate lysis buffer. Need to detect p66 isoform. | NaAc/SC rem. | 8% Tris-Glycine | Shc-mo: 1:1000 (upstate #06-203). HRP-conj-anti-Mo 1:1500. | 20mg gave 591µg protein (yield of 3%). 65mg gave 1622 µg (yield of 2.5%) | - | - |
| 020808 | MAL/EED | Ordering new materials | Amersham Biosciences: Full range rainbow MW marker RPN800; precut 7X8cm Hybond ECL membranes RPN78D; ECL western blot detection system reagents RPN2108. ECL Hyperfilm 18X24cm RPN2103. | New marker works nicely. | Use new marker | ||||
| 020812 | MAL/EED | Sc & muscle p75 | Comparing SC and muscle p75 signals. Loaded amount: 20, 40 and 80 µg protein. | NaAc/SC&Mu | 8% Tris-Glycine | p75-rb 1:10000 (gift Wesskamp)
HRP-conj-anti-Rb 1:1500 |
- | - | - |
| 020812 | MAL/EED | Ordering new gels | Novex precast Tris-Glycine gels: 12%, 1.5mm, 15well, EC60085. | - | - | - | - | - | - |
| 020814 | MAL/EED | anti-Shc (pan) | Comparing SC and muscle Shc signals. Loaded amount: 20, 40 and 80 µg protein. | NaAc/SC & Mu | 8% Tris-Glycine | Shc-mo: 1:1000 (upstate #06-203). HRP-conj-anti-Mo 1:1500. | - | - | - |
| 020819 | MAL/EED | Molding gels | 10% Tris-glycine gels. Agarose 1% used to seal gel casettes. | - | 10% Tris-Glycine | - | - | - | Excellent! |
| 020820 | MAL/EED | Testing ED2 on home-made gels | Loaded amount: 20, 40 and 80 µg protein. ED2 is constitutively expressed in MØ. | NaAc/Mu | 10% Tris-Glycine | ED2-mo 1:5000 (Serotec 1999).HRP-conj-anti-Mo 1:1500. | Clear bands, probably specific but size does not match marker. Likely due to samples not being reduced while reducing agent included in marker. | - | - |
| 020821 | MAL/EED | Discovering method error | No reducing agent included in sample buffer | - | - | - | - | Unreduced samples will not migrate correctly during electrophoresis. | ß-Mercapto-Ethanol should be added to 5% in sample buffer (2X). Final concentration 2.5% when loaded. |
| 020821 | MAL/EED | Ordering Shc antibodies | Ordering Shc pan and p66 specific antibodies (Rb-Shc-mo: Upstate #06-203) and Rb-p66 Upstate #07-150) via KeLab. | - | Rb-Shc: 1:1000
Rb-p66: Positive control included. |
- | - | - | |
| 020826 | MAL/EED | Extracting protein from muscle D11, 15, 18, 28 and yad B1-5 | Protein extracted from Gastroc muscle of D11: 58; D15: 88; D18: 70; D28: 75mg and yad B1: 39; B2: 50; B4: 30; B5: 36mg. Protein extracted in 500µl NaAcetate lysis buffer. Spinning after inc: 25min at max speed 13K G. Aliquots of 5µg/µl prepared and stored in -70ºC upright freezer. | NaAc/Mu | - | - | Supernate volumes all about 450µl. | - | - |
| 020902 | MAL/EED | Testing p66 spec AB | Muscle, SC and positive control samples | NaAc/ Mu, SC, PC (10µg) | 4-12% wide range gel | Rb-p66 Upstate #07-150 | - | - | - |
| 020903 | MAL/EED | Shc-p66 aliquots prepared | Upstate Rb-p66 #07-150 antibody aliquoted 5µl/tube. Suggested dilution 1:1000-1:2000. Stored in upright -70ºC freezer. | - | - | Rb-p66 Upstate #07-150 | - | - | - |
| 020906 | MAL/EED | ShcA-pan muscle | Mu D18,11,28, Yad + positive control
10, 20, 40 µg protein per lane and animal |
NaAc/Mu | 4-12% wide range gel | PrAb: Rb-Shc: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
- | - | - |
| 020911 | MAL/EED | p66-ShcA muscle | Mu D18,11,28, Yad + positive control
10, 20, 40 µg protein per lane and animal |
NaAc/Mu | 4-12% wide range gel | PrAb: Rb-p66: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
- | - | p66 antibody does not work very well in Mu, gives a lot of background. optimize? |
| 020912 | MAL/EED | p66-ShcA SC | SC-Th D18, 11, 28 and yad
10, 20, 40 µg protein per lane and animal |
NaAc/Sc | 4-12% wide range gel | PrAb: Rb-p66: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
- | - | p66 antibody does not work very well in Sc, gives a lot of background. optimize? |
| 020913 | MAL/EED | ShcA-pan SC
two experiments |
SC-Th D18, 11, 28 and yad
10, 20, 40 µg protein per lane and animal |
NaAc/Sc | 4-12% wide range gel (2 gels) | PrAb: Rb-Shc: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
Gel 1 and 2 looks the same, D11 shows strange results | - | - |
| 020917 | MAL/EED | ShcA-pan muscle | Gastroc muscle D15,18, 11, 28 and yad
10, 20, 40 µg protein per lane and animal |
NaAc/Mu | 4-12% wide range gel | PrAb: Rb-Shc: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
- | - | - |
| 020918 | MAL/EED | ShcA-pan SC | SC-Th D15,18, 11, 28 and yad
10, 20, 40 µg protein per lane and animal |
NaAc/Sc | 4-12% wide range gel | PrAb: Rb-Shc: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
D11 is still not good | - | - |
| 020924 | MAL/EED | ShcA-pan SC | SC-Th D15,18, 11, 28 and yad.
10, 20, 40 µg protein per lane and animal |
NaAc/Sc | 4-12% wide range gel | PrAb: Rb-Shc: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
D11 is still not good | - | - |
| 020926 | MAL/EED | ShcA-pan SC | SC-Th D15,18, 11, 28 pooled and yad with positive
control
10, 20, 40 µg protein per lane and pooled animals and Yad |
NaAc/Sc | 10% Tris-Glycine self-made | PrAb: Rb-Shc: 1:1000
SecAb: HRP-conj-anti-Rb 1:1500. |
- | - | - |
| 021001 | MAL/EED | Testing IGF-1 antibodies | Mu, test tissue (m.gastroc)
25, 50 and 100 µg protein per lane. |
NaAc/Mu | 15% Tris-Glycine self-made | Rb-anti-IGF-1 Peninsula labs # IHC7308. Dil: 1:10 000. Detection HRP-conj-anti-Rb 1:1500.Mo-anti-IGF-1 Upstate #05-172. Sugg. Dil: 1µg/1000µl. From aliquot dil: 1:10 000. Detection HRP-conj-anti-Mo 1:1500. | Polyclonal shows a lot of large bands. 40% Cross-R
with IGF-2.
Monoclonal shows few and distinct bands. Repeat experiment needed to confirm correctness of sizes (ladder needs to be cut away before blocking). Compare bands at 14 (IGF-1) and 12KD (IGF-2) |
-Smiley effect on gel phoresis. Agarose sealing is liquid proof but not electrophoretically inert. | Very nice separation in 15% gel. Polyclonal good for identifying IGF-2 in comparison with IGF-1 mono. Extra bands would be nice to confirm as IGF-1 complexed to bp:s. |
| 0210ZZ | EED | Re-testing IGF-1 antibodies | Mu, test tissue (m.gastroc)
25, 50 and 100 µg protein per lane. |
NaAc/Mu | 15% Tris-Glycine self-made | Rb-anti-IGF-1 Peninsula labs # IHC7308. Dil: 1:10 000. Detection HRP-conj-anti-Rb 1:1500.Mo-anti-IGF-1 Upstate #05-172. Sugg. Dil: 1µg/1000µl. From aliquot dil: 1:10 000. Detection HRP-conj-anti-Mo 1:1500. | No results gel run useless. | Migration uneven, heading for side of gel. | Agarose does not block electrolyte movement! |
| 021025 | EVI | Ordering new gel casettes | - | - | - | 1.0mm, 1.5mm and 12 lane 1mm combs. | - | - | - |
| 021107
021108 |
EED | Testing IL-6 & retesting IGF-1 antibodies
run+rerun |
Mu, test tissue (m.gastroc)
40, 80 µg protein per lane. |
NaAc/Mu | 15% Tris-Glycine self-made | Goat-anti-IL-6 Santa Cruz #R19, dil 1:1000. Detection Jackson HRP-conj-anti-Go 1:2000. Rb-anti-IGF-1 Peninsula labs # IHC7308. Dil: 1:10 000. Detection HRP-conj-anti-Rb 1:2000.Mo-anti-IGF-1 Upstate #05-172. Sugg. Dil: 1µg/1000µl. From aliquot dil: 1:10 000. Detection HRP-conj-anti-Mo 1:2000. | - | First run cover left on membrane = no transfer. Second run some of bubbles at transfer. Primary stayed on for too long (2.5h) and backgrounf was immense for the polyclonals but not for the monoclonal. | - |
| 021111 | EED | Retesting IL-6, testing CNTF and FGF-2 | Mu, test tissue (m.gastroc)
40, 80 µg protein per lane. |
NaAc/Mu | 15% Tris-Glycine self-made | Goat-anti-IL-6 Santa Cruz #R19, dil 1:1000. Detection Jackson HRP-conj-anti-Go 1:1500. CNTF Chemicon #MAB338 | - | - | We need to prepare more test lysates. |
| 021112 | EED | Testing CNTF and LIF | Mu, test tissue (m.gastroc)
25, 50 µg protein per lane. |
NaAc/Mu | 15% Tris-Glycine self-made | CNTF Chemicon #MAB338 mo 1:5000/10000; Detection HRP-conj-anti-Mo 1:2000 LIF polyclonal (TH-lab); Detection HRP-conj-anti-Rb 1:5000 | - | - | - |
| 021113 | EED | IGF-1 mo | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | Mo-anti-IGF-1 Upstate #05-172. Sugg. Dil: 1µg/1000µl. From aliquot dil: 1:10 000. Detection HRP-conj-anti-Mo 1:2000. | - | - | - |
| 021114 | EED | IGF-1 Rb | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | Rb-anti-IGF-1 Peninsula labs # IHC7308. Dil: 1:10 000. Detection HRP-conj-anti-Rb 1:2000 | ok | - | - |
| 021115 | EED | CNTF mo | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | CNTF Chemicon #MAB338 mo 1:10000. Detection HRP-conj-anti-Mo 1:2000 | Strange results: reverse migration in some lanes? | - | - |
| 021217 | EED | IGF-1 Rb rerun | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | Rb-anti-IGF-1 Peninsula labs # IHC7308. Dil: 1:10 000. Detection HRP-conj-anti-Rb 1:2000 | Not ok | Strange result! some samples have reduced total signal -includes background | - |
| 030113 | EED | IGF-1 Rb rerun | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | Rb-anti-IGF-1 Peninsula labs # IHC7308. Dil: 1:10 000. Detection HRP-conj-anti-Rb 1:2000 | ok | - | - |
| 030114 | EED | IGF-1 Rb rerun | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | Rb-anti-IGF-1 Peninsula labs # IHC7308. Dil: 1:10 000. Detection HRP-conj-anti-Rb 1:2000 | ok | - | - |
| 030114 | EED | CNTF mo | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | CNTF Chemicon #MAB338 mo 1:10000. Detection HRP-conj-anti-Mo 1:2000 | Error in loading exp not completed | - | - |
| 030129 | EED | CNTF mo | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate | NaAc/Mu | 15% Tris-Glycine self-made | CNTF Chemicon #MAB338 mo 1:10000. Detection HRP-conj-anti-Mo 1:2000 | Nice results! | - | - |
| 030130 | EED | CNTF mo & LIF rb | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. Testing 2ml antibody volume in laminated plastic sheath. | NaAc/Mu | 15% Tris-Glycine self-made | CNTF Chemicon #MAB338 mo 1:10000. Detection HRP-conj-anti-Mo 1:2000. LIF polyclonal 1:10000 Detection HRP-conj-anti-Rb 1:2000 | CNTF -as before.
LIF weak bands but looks ok. |
Laminating device tricky, sometimes melts, sometimes seals, never perfect! | Reuse antibody instead, as suggested by G. Schulte: dilute in buffer + 5%BSA and add NaAz; can be reused several times... |
| 030206 | MAL/EED | Preparing protocols for phospho-western | New extraction protocol: TRIS-base buffer including protease inhibitors and phosphorylation stabilizers. | - | - | - | - | - | - |
| 030207 | MAL/EED | Protein extraction using phospo-protocol | Test tissue: wistar, female 020729, mu gastroc. TRIS-base protocol. | TRIS-(p)/Mu | - | - | 6X50µl 33.35µg/µl (yield 21.3 µg/mg) | - | - |
| 030210 | MAL/EED | Test: phospho-Akt | Phospho-spcific antibody p-Akt tested on mu gastroc test tissue extracted 030207. 300, 200, 100, 50µg loaded. | TRIS-(p)/Mu | 10% Tris-Glycine self-made | p-Akt (Ser473) Cell Signaling #9271 Rb1:1000
Detection HRP-conj-anti-Rb 1:1500 |
somewhat weak signal: 15min exposure ok but far from saturated | - | - |
| 030212 | EED | CNTF mo & LIF rb | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. Lamination was quite messy; will try to dissolve LIF-ab in PBS containing BSA instead of milk and add some NaAz which should allow reuse of ab. | NaAc/Mu | 15% Tris-Glycine self-made | CNTF Chemicon #MAB338 mo 1:10000. Detection HRP-conj-anti-Mo 1:2000. LIF polyclonal 1:10000 Detection HRP-conj-anti-Rb 1:2000 | Gel ruptured before transfer; used for CNTF confirmation run. | Weak signals but ok. | - |
| 030218 | EED | LIF rb & IL-6 Go | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. Protocol change: 45min room temp blocking and overnight primary Ab incubation | NaAc/Mu | 15% Tris-Glycine self-made | LIF polyclonal 1:10000 Detection HRP-conj-anti-Rb 1:2000. Goat-anti-IL-6 Santa Cruz #R19, dil 1:4000. Detection Jackson HRP-conj-anti-Go 1:2000. | - | Weak LIF-signals but ok result. No specific IL-6 bands | Overnight incubation with primary not a problem nor an advantage. 45min blocking at RT works -compress protocol..? |
| 030227 | EED | IL-6 & bFGF test. | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. Protocol change: 45min room temp blocking and overnight primary Ab incubation | NaAc/Mu | 15% Tris-Glycine self-made | Goat-anti-IL-6 Santa Cruz #R19, dil 1:4000. Detection Jackson HRP-conj-anti-Go 1:2000. bFGF () from S.K.s IHC dilutions. Dil: 1:4000. Detection HRP-conj-anti-Rb 1:2000. | - | - | - |
| 030301 | EED | IL-6 | Mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. Protocol change: 45min room temp blocking and overnight primary Ab incubation | NaAc/Mu | 15% Tris-Glycine self-made | Goat-anti-IL-6 Santa Cruz #R19, dil 1:4000. Detection Jackson HRP-conj-anti-Go 1:2000. | - | messy membrane but results discernable | Insufficient wash after primary? |
| 030328 | EED | IL-6 rerun, bFGF and MyoD-test | Weekend prep:
samples for new bFGF antibody test, MyoD-test and new IL-6 run |
NaAc/Mu | 15% Tris-Glycine self-made | - | - | - | - |
| 030329 | EED | IL-6 rerun, bFGF and MyoD-test | IL-6 rerun mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. bFGF and MyoD-test on yad mu lysate (+D15 for myoD) | NaAc/Mu | 15% Tris-Glycine self-made | Goat-anti-IL-6 Santa Cruz #R19, dil 1:4000. Detection Jackson HRP-conj-anti-Go 1:2000. Mo-anti-bFGF (Upstate #05-118). Dil: 1:2000. Detection HRP-conj-anti-mo 1:2000. Mo-anti-MyoD1 (Novocastra) dilutions. Dil: 1:500. Detection HRP-conj-anti-mo 1:2000. | Difficult to judge size of bands; sizes above 30 not distinguishable | Too quick phoresis -wrong gel conc (Zheng-prep)? Separation between 10-20(30) Several small size bands from bFGF. IL-6 still alot of black smearing. | Both bFGF and MyoD seem to work. MyoD only one band of approx correct size. Test decreased conc of anti-goat secondary, 1:4000? |
| 030331 | EED | LIF rb rerun | LIF rerun mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. | NaAc/Mu | 15% Tris-Glycine self-made | 1:4000. LIF polyclonal 1:10000 Detection HRP-conj-anti-Rb 1:2000. | - | All bands visible, except lowermost two bands, are within correct range 38-67 | Super: LIF done X3 |
| 030401 | EED | bFGF first run and Zheng retests antibodies IL-6 and MyoD | bFGF run mu gastroc, D15,18,11,28 on 25 & 50 µg protein lysate. For IL-6 retest: try more diluted secondary and blocking + secondary using combination block milk +BSA. Note: due to confusion a mix of secondary anti-mo/go was used for MyoD and IL-6. | NaAc/Mu | 15% Tris-Glycine self-made | Mo-anti-bFGF (Upstate #05-118). Dil: 1:2000.
Detection HRP-conj-anti-mo 1:2000.
Goat-anti-IL-6 Santa Cruz #R19, dil 1:4000. Detection Jackson HRP-conj-anti-Go 1:4000. Mo-anti-MyoD1 (Novocastra) dilutions. Dil: 1:500. Detection HRP-conj-anti-mo 1:2000. |
bFGF: Specific bands strong signal but some bands
of incorrect size.
IL-6 and MyoD1 almost identical spread of multiple bands and high background. |
- | Secondary Ab anti goat is the problem: background + unspecific bands! |
| 030401 | EED | bFGF rerun | - | NaAc/Mu | 15% Tris-Glycine self-made | - | Confirms previous result | - | - |
| 030403 | Zheng | Test: MyoD1 and secondaries anti-mo, anti-rb | Test: MyoD and secondary antibodies (Amersham) anti-mo, anti-rb 1:2000 (1.5h) after overnight incubation with 5%BSA in PBS but no primary. Test protein: wistar rat lysate | NaAc/Mu | 15% Tris-Glycine self-made | Mo-anti-MyoD1 (Novocastra) dilutions. Dil:
1:500. Detection HRP-conj-anti-mo 1:2000.
Secondary test: HRP-conj-anti-mo 1:2000. HRP-conj-anti-rb 1:2000. |
- | - | - |
| 030701-03 | MAL/EED | Test of Antibodies:
P-Akt, Akt, P-Shc, Shc |
Mu exctracted with Tris-buffer and protease inhibitor.
50, 100 & 200µg lysate. For Akt Positive and negative controls
were used (Cellsignaling
#9273) and Positive control for Shc (Upstate
#12-301)
Block 1h (PBS + 5%milk + 0.1% Tween20) Primary overnight (PBS + 5%BSA + 0.1% Tween20) Secondary 1h (PBS + 5%milk + 0.1% Tween20) |
TRIS-(p)/Mu | 10% Tris-Glycine self-made | Primary Antibodies:
Rb-anti-Shc (Upstate #06-203) 1:000 Rb-anti-P-Shc (Tyr317) (Cellsignaling #2431) 1:1000 Rb-anti-Akt (Cell Signaling #9272) 1:1000 Rb-anti-P-Akt (Ser473) (Cell Signaling #9271) 1:1000 Secondary Antibody: HRP-conj-anti-Rb 1:2000 for all |
Shc, Akt and P-Akt seemd to work, P-Shc did not produce any bands | - | - |
| 031006 | EED | Retesting MyoD & IGF-I (mo) | Test protein: wistar rat lysate and positive control
for Shc (10µ) (Upstate
#12-301). Negative control =primary omitted.
Protocol: 35min 5% milk block, Wx3, Prim 1h; Wx3, Sec 1h, Wx3. ECL plus used first time. ca 5ml is sufficient to cover membrane (5min incub.). |
NaAc/Mu | 15% Tris-Glycine self-made | Mo-anti-MyoD1 (Novocastra) dilutions. Dil:
1:500. Detection HRP-conj-anti-mo 1:1000.
Mo-anti-IGF-1 Upstate #05-172. dil: 1:1000. Detection HRP-conj-anti-Mo 1:1000 |
Neg control reveals bands very similar to both IGF-I and MyoD1. | Next trial: Dilute secondary more. Try tween-20 instead of Triton-X100 in buffers. Increase myoD1 conc (rec IHC dil =1:80). | ECL plus -easy to use, good signal. |
| 031007 | EED | Retesting MyoD & IGF-I (mo) | Test protein: wistar rat lysate. Negative control =primary omitted | NaAc/Mu | 15% Tris-Glycine self-made | Mo-anti-MyoD1 (Novocastra) dilutions. Dil:
1:500. Detection HRP-conj-anti-mo 1:2000.
Mo-anti-IGF-1 Upstate #05-172. dil: 1:1000. Detection HRP-conj-anti-Mo 1:2000 |
Neg control reveals bands very similar to both IGF-I and MyoD1. | ||
| 031008 | EED | Preparing new muscle and liver test/control lysates | Preparing new muscle and liver test/control lysates | RIPA
NP40, NaDeoxyChol |
- | ||||
| 031010 | EED | Retesting IGF-I (Rb) | Test protein: wistar rat lysates, SD liver. Negative control =primary omitted | NaAc/Mu
RIPA/mu/liv |
15% Tris-Glycine self-made | Mo-anti-IGF-1 Upstate #05-172. dil: 1:1000. Detection HRP-conj-anti-Mo 1:2000 | |||
