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Western blotting: p53 very faint, Bax and BCl-2 also wish cells

Western blotting: p53 very faint, Bax and BCl-2 also wish cells - General Science Questions and Layperson Board

Western blotting: p53 very faint, Bax and BCl-2 also wish cells - General Science Questions and Layperson Board


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Old 04-24-2012, 08:14 PM
Pipette Filler
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Angry Western blotting: p53 very faint, Bax and BCl-2 also wish cells



Hi everybody,
I am doing western blot using wish cell lines. I am getting good result for GAPDH and caspase 3. But the bands for p53, Bax and BCl-2 are very faint?!

Using the following protocol. SDS page using 10% resolving gel, Using santa cruz Ab primary Ab diluted to 1:500 and secondary to 1:5000. Finally developing blots using TMBH2O2. For GAPDH protocol even works at 1:1000.





Sample preparation and SDS gel electrophoresis:
• Harvest fully grown cells (from 25cm2 culture flask) after washing with 1X PBS.
• Lyse cells in 500 ΅l pre-cooled lysis buffer (62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT) on ice for 20 min.
• Centrifugation at 5000 rpm in a bench top for 5 min.
• Use supernatant
• 25 ΅g protein were used for electrophoresis.
• Protein loading dye (6x) were added in protein samples and denatured at 95-100 °C for 5 min before electrophoresis.
• Protein storage: Protein samples can be stored at room temperature up to 6 weeks. For long-term storage, keep it at 4°C. [Sodium azide (NaN3) at a final concentration of 0.02-0.05% (w/v) antimicrobial]
Protein estimation using Lowry protein assay

Reagents and instruments required:
1. Folin Ciocalteu Reagent: Folin - Ciocalteau reagent solution (1N) Dilute commercial reagent (2N) with an equal volume of water on the day of use (2 ml of commercial reagent + 2 ml distilled water).

2. Analytical reagent: [Add 2 g Sodium carbonate and 0.4 g NaOH per 100 ml of distilled water. Remove 2 ml from this 100 ml add 1 ml of sodium potassium tartarate (2% Solution) and 1 ml of CuSO4 solution (1%)].

3. BSA stock solution (1mg/ml): Prepare 1 ml of different dilutions of BSA from stock solution such as 0.5mg/ml, 0.25 mg/ml, and 0.125mg/ml by diluting the stock in water.

4. Laboratory grade or distilled (DI) water

5. Spectrophotometer and cuvettes or microplate reader and microplate


Protocol

• Mix 100 ΅l of each standard with 900 ΅l l of water in 15 ml corning tubes.
• Add 20 ΅l of sample to be analyzed with 980 ΅l of water in 15 ml corning tubes.
• Add 5 ml of analytical reagent prepared above in each sample and standard tubes.
• Mix well by vortexing
• Incubate at room temperature for 10 min.
• Add 0.5 ml of working Folin’s solution and mix well
• Incubate the tubes at room temperature for additional 30 min.
• Read the OD (of blue/violet colour developed) at 660nm.








Results:
----------------------------------------------------------------------------------------------------------------
Sample Conc Abs (650 nm)
Standard 0.125 0.193
Standard 0.25 0.308
Standard 0.5 0.483
Standard 1 0.885
Sample (5 times) Unknown 0.198


Y=0.7825X + 0.1004; R2= 0.9991
We can calculate the protein concentration (x) using above equation by putting the OD660 (Y) of the sample and the dilution factor.
X = (0.198-0.1004)/0.7825*5*5= 3.12 mg/ml.
---------------------------------------------------------------------------------------------------------------------------
SDS Page analysis Reagents
1. Acrylamide: Bis-Acrylamide mix -100 ml: Dissolve 29g acrylamide and 1g N, N’-methylenebisacrylamide in 60 ml dH2O. Heat the solution at 37 0C; Adjust the volume to 100ml
2. For 50 ml of 6X loading buffer: Tris (pH 6.8) [final conc 50mM] for 6X add 10 ml of 1.5 M to 20 ml; SDS [final conc 2%] add 6g; β-Mercaptoethanol [final conc 100mM] add 1.59 ml of 14.7 M (in bottle); Glycerol 30 ml; Bromophenol blue 200 mg.
For 10 ml of 4X loading buffer: 2.0 ml of 1M Tris-HCl (pH 6.8), 0.8 g SDS, 4.0 ml 100% glycerol, 0.4 ml of 14.7 M β-mercaptoethanol, 1.0 ml 0.5M EDTA, 8.0 mg BPB adjust vol with dH20 to 10 ml.
3. Tris 1.5 M (pH=8.8): Tris= 18.1gm; adjust pH with HCl to 8.8; Mix well, adjust the pH=8.8 and make the final volume with 100ml dH2O
4. Tris 1.5 M (pH=6.8): Tris= 18.1gm; Adjust pH=6.8 with HCl and make the final volume 100ml dH2O.
5. Ammonium persulphate (APS): 1gm APS dissolves in 10ml dH2O. Make aliquots and keep it at -20oC.
6. Sodium Lauryl Sulphate (SDS): 1gm SDS dissolves in 10ml dH2O.
7. 10X Running Buffer (pH=8.3): Dissolve 10g SDS, 30.3g Tris and 144.1g glycin in 800ml H2O. Adjust the pH=8.3 and make final volume up to 1000ml in dH2O. Store at room temperature
8. Staining Solution: Coomassie Brilliant blue R-250= 0.25%; Methanol= 45%; Glacial Acetic acid= 10%; adjust with dH2O
9. De-staining Solution: Methanol= 45%; Glacial Acetic acid= 10% in dH2O

For casting gel:

• Add 5 ml of resolving gel solution into the gel cast wait 1h
• After one hour remove water from the surface of polymerized gel using a tissue paper.
• Now add stacking gel solution upto the top of the gel plate insert comb and wait for another 30 min-1hr.
• Electrophoresis in 1X running buffer (pH 8.8; prepared by diluting 10X buffer described above was carried out with 25 mA/gel.
• After electrophoresis one of the gel was stained with staining solution for 30 min-1 hr.
• And was de-stained with de-staining solution
• Another gel was used for blotting
Blotting
Solutions and Reagents
NOTE: Prepare solutions with Milli-Q or equivalently purified water.
1. 1X Phosphate Buffered Saline (PBS).
2. 1X SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT.
3. Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5) Optional 0.05% SDS.
4. 10X Tris Buffered Saline (TBS): To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X).
5. Nonfat Dry Milk or BSA (bovine serum albumin): 5% weight to volume [w/v].
6. Blocking Buffer: 1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk/BSA; for 150 ml, add 15 ml 10X TBS to 135 ml water, mix. Add 7.5 g nonfat dry milk/BSA and mix well. While stirring, add 0.15 ml Tween-20 (100%).
7. Wash Buffer: 1X TBS, 0.1% Tween-20 (TBS/T).
8. Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% BSA; for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g BSA and mix well. While stirring, add 20 μl Tween-20 (100%).
9. Primary antibody for β-actin (Santa Cruz SC-130657 rabbit)
10. Secondary antibody Goat anti rabbit : dilute in 1X TBST (Santa Cruz SC-2004).
11. Prestained Protein Marker (Fermentas: pageRuler #SM0671)
12. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes, which CST recommends. PVDF membranes may also be used.
B. Protein Blotting
• Submerge the membrane (Specifications provided above) in methanol.
• Pre-wet the pads and the filters in 1X transfer buffer.
• Prepare the sandwich of pads, filter papers, gel, memberane, filter paper and pads as shown in figure below.
• Protein moves from anode (-) black to cathode (+) red so the gel should be on the negative side and the membrane on the positive side.
• Electrotransfer to PVDF membrane (Milipore; Immobilon cat No. IPVH20200). Use 1 transfer buffer. Current 250 mA for 2 hrs at room temperature or 130 mA overnight.
D. Membrane Blocking and Antibody Incubations
• NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
• (Optional) After transfer, wash membrane with 25 ml TBS for 5 minutes at room temperature.
• Incubate membrane in 25 ml of blocking buffer for (O/N in our case) 1 hour at room temperature.
• Wash three times for 5 minutes each with 15 ml of TBS/T.
• Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
• Wash three times for 5 minutes each with 15 ml of TBS/T.
• Incubate membrane with appropriate HRP-conjugated secondary antibody (1:2000) in 10 ml of blocking buffer with gentle agitation for 1 hour at room temperature.
• Wash three times for 5 minutes each with 15 ml of TBS/T.
• Detection of Proteins
• Incubate membrane with 2 ml of 1(X) TMBT/H2O2 [GeNeiTM]for 30 min.

Results: As non specific binding was observed.
Experiment was repeated with following modification
1. Primary Ab was diluted this time 1:1000 times
2. Exposure to Primary Ab was ON
3. Secondary Ab was diluted to 1:5000
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bax , bcl-2 , bcl2 , blotting , cells , faint , p53 , western , western blotting: western , wish cells


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