Experimental Procedure of Primary Antibody

Protocol for Immunoblotting and Immunoprecipitation:

Preparation of Cell Lysate:

Add RIPA buffer to cells(100μl to a 35mm dish, 200μl to a 60 mm dish, 500 uL to a 100 mm dish) while the culture dish is placed on ice. Scrape the cells and gently rock the suspension on either a rocker or an orbital shaker in the cold room for 15 minutes to lyse cells. Sonicate in ice water with bath sonicator, until the sample is no longer viscous. Centrifuge the cells at 12000 g at 4°C for 5 minutes to remove pellet. Move the supernatant to a fresh tube. Final concentration of cell lysate will be 2-3 ug/uL. Add 5 x SDS stop buffer to the lysate to a 1xSDS final concentration .

Immunoblotting:

1. After boiling for 10 minutes, 50-80 uL sample will be loaded to SDS PAGE. For most proteins, 100-200 ug total lysate proteins per lane loading amount is suggested, because 80% of protein species in cells are at very low concentration.
2. Soak the gel in western blot transfer buffer. Cut a piece of membrane to the size of the gel. PVDF membrane is recommended for most proteins. Dip the membrane into methanol for 1-2 minutes, soak the membrane in transfer buffer for 10 minutes, and place it on a thick stack of buffer-soaked filter paper. Then cover it with another stack of buffer soaked filter papers. Cover up the transfer apparatus. Gel should be on the negative side of the membrane.
3. Run for 90 minutes at current of 1 mA per cm² .
4. Incubate the membrane in blocking buffer for 1 hour at room temperature or overnight at 4°C. Wash membrane 3x5 min with washing buffer.
5. Dilute primary antibody with blocking buffer. Incubate the membrane for 1 hour at room temperature or overnight at 4°C. Wash membrane 3x5 min with washing buffer.
6. Dilute secondary antibody with blocking buffer. Incubate the membrane for 1 hour at room temperature. Wash membrane 3x5 min with washing buffer.
7. Wash membrane 3x10 min with washing buffer.
8. Develop color with ECL.

Immunoprecipitation:

1. 1. Prepare cell lysates or other protein samples. Cellular proteins or other protein samples should be labeled either metabolically or by iodination or biotination. For cell lysate, a concentration of 1-2 ug/uL protein is optimum.
2. To 500 ug - 1 mg cell lysate, add 50 uL of 50% slurry of protein A-sepharose beads and incubate the mix at 4°C for 15 minutes. Centrifuge at 10000 g for 5 minutes at 4 °C. Collect the supernatant.
3. To the supernatant add 5 ug of rabbit polyclonal antibodies against the specific antigen. Gently rock the reaction mixture for 1 hour at 4 °C.
4. Add 50 uL of 50% slurry of protein A-Sepharose beads to the reaction mixture and gently rock the sample at 4°C for 30 minutes.
5. Spin the sample at 10000 g at 4°C for 3 minutes to pellet the beads.
6. Wash the beads twice with washing buffer A (10 mM Tris-HCl, pH 8, 500 mM NaCl, 0.5% NP-40 and 0.05% SDS), once with washing buffer B (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% NP-40, 0.05% SDS and 0.5% Dideoxycholate), once with washing buffer C (10 mM Tris-HCl, pH 8, 0.05% SDS).
7. Add 100 uL of 1.25 x SDS loading buffer and boil the sample for 10 minutes before loading to SDS PAGE.
8. Detect the immunoprecipiated sample on SDS-PAGE either by direct radioautography or by further blotting biotin, depending on the initial labeling method.

Protocol for Immunofluorescence with Tissue Culture Cells

1. Culture the cells on small round cover glass.
2. Fix cells with formalin-PBS (10%) for 10 minutes at 4°C.
3. Transfer the glass into pre-chilled acetone (-20°C) for exact 5 minutes.
4. Transfer the cover glass into PBS, the cells on cover glass can be stored in PBS-NaN3 for several months at 4°C. 5. Block the cells with BSA-PBS-NaN₃.
5. Add 40 uL of specific primary antibodies at concentration of 1 ug/mL on cover glass and incubate for 1 hour at room temperature in a moist container in dark.
6. Wash the cover glass with 6 cups of PBS-N aN₃.
7. Add 40 uL of fluorescence-labeled secondary antibody on each cover glass and incubate for 1 hour at room temperature in a moist container in dark.
8. Mount the coverslip in 15% glycerol or a small volume of hydromount on a slide.
9. Seal the coverslip with nail polish on the edge.

Protocol for Immunofluorescence with frozen tissue section

1. The frozen sections were removed and balanced at room temperature for 10-20min. The sections could not be completely dried.
2. Fix with 4% paraformaldehyde at room temperature for 15min, then rinse 3 x 5 min with TBS.
3. Place section in vessel filled with 3% H₂O₂ solution at room temperature for 10min. The solution must be prepared freshly .The preparation method of 3% H₂O₂ solution is to add 1ml 30% H₂O₂ to 9ml methanol, mix completely.
4. Rinse 3 x 5 min with TBS.
5. Block in 5% BSA solution for 30 minutes at 37℃.
6. Drain slides for a few seconds (do NOT rinse) and wipe round sections.
7. Add 40 uL of specific primary antibodies at concentration of 1 ug/mL on cover glass and incubate for 2 hour at 37℃ or overnignt at 4℃ in a moist container in dark.
8. Rinse 1 x 5 min with TBST. Then rinse 3 x 5 min with TBS.
9. Add 40 uL of fluorescence-labeled secondary antibody on each cover glass and incubate for 1 hour at room temperature in a moist container in dark.
10. Rinse 1 x 5 min with TBST. Then rinse 3 x 5 min with TBS.
11. Add DAPI or PI working solution to cover glass and incubate for 10min at room temperature in dark.
12. Rinse 3 x 3 min with TBS.
13. Mount the coverslip in 15% glycerol or a small volume of hydromount on a slide.
14. Seal the coverslip with nail polish on the edge.

Protocol for Immunohistochemistry on Paraffin-Embedded Tissue Sections

1. Deparaffinize slides in 2 changes of xylene, one for 40 minutes, the other for 20 minutes.
2. Transfer slides to 100% alcohol, 95% alcohol, 80% alcohol, 60% alcohol and ddH₂O for 5 minutes each.
3. Place slides in vessel filled with antigen retrieval buffer and microwave on middle for about five minutes (700 W oven), allow retrieval solution to cool at room temperature.
4. Rinse 2 x 5 min with TBS.
5. Block endogenous peroxidase activity by incubating sections in 3% H₂O₂ solution for 30 minutes.
6. Rinse 2 x 5 min with TBS.
7. Block in 5-10% goat serum in TBS for 30 minutes at room temperature.
8. Drain slides for a few seconds (do NOT rinse) and wipe round sections.
9. Apply primary antibody made up in TBS. Incubate 1 hour at room temperature or 4°C overnight.
10. Rinse 2 x 5 min with TBST.
11. Apply secondary biotinylated antibody made up in TBS for 1 hour at room temperature.
12. Rinse 2 x 5 min with TBS.
13. Apply streptavidin peroxidase for 15 minutes or 30 minutes at room temperature.
14. Rinse 2 x 5 min with TBS.
15. Develop with chromogen (DAB) at room temperature, watching under microscope.
16. Rinse in running tap water for 5 minutes.
17. Counterstain in mayor’s hematoxylin bath for 30-60 seconds.
18. Wash in water bath 7-8 times, then tap water for 3 minutes.
19. Dehydrate through 60%, 80%, 95% and 100% alcohol for 5 minutes each. 20. Transfer to xylene for 5 minutes. Air for 30 minutes.
20. Mounting.

Buffers

10 x PBS 2000 mL Transfer buffer 1000 mL BSA-PBS-NaN3 1000 mL

H2O 1000 mL Tris base 48 mM BSA 5 g

KCl 4 g Glycine 39 mM 1 x PBS-Azide 1000 mL

KH2PO4 4 g SDS 0.037%

Na2HPO4· H2O 22.9 g Methanol 20%

NaCl 160.0 g

ddH2O up to 2 liter,

Filter and Autoclave

Washing buffer TBST pH 7.4 RIPA buffer 1000 mL Blocking buffer

10 mM Tris-HCl Sodium Chloride 150 mM Non-fat Milk 5 g

100 mM NaCl NP-40 or Triton X-100 1.0% Washing buffer TBST 100mL

0.2% Tween-20 Sodium Deoxycholate 0.5%

SDS 0.1% PBS-NaN3 1000 mL

TBS-Triton X-100 1000 mL Tris base 50 Mm 1 x PBS 1000 mL

TBS 1000 mL EGTA 1 mM Na-Azide 2 g

Triton X-100 250 uL EDTA 5 mM

Sodium Fluoride 5 -10 mM

Sodium Orthovanadate 1 mM

Antigen retrieval buffer 500 mL TBS 1000 mL

0.1 M Citric Acid 9 mL Tris base 3 g

0.1 M Sodium Citrate 41 mL NaCl 8 g

ddH2O 450 mL ddH2O 1000 mL

pH 6.0 Adjust pH to 7.6 with HCl

Immunohistochemistry Q&A

Technically, IHC and ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be identified and optimized for each individual IHC/ICC study. Optimization of IHC/ICC may also require troubleshooting a variety of factors. The tables below highlight common IHC/ICC issues and provide appropriate experimental actions. For further assistance, please contact our technical service department.

Problem: Lack of Staining

Problem: High Background

Problem: Cell/Tissue Morphology is Destroyed

Problem: Staining is Inappropriate

Western Blot Q&A

For further assistance, please contact our technical service department.

Problem: No Bands Observed

Problem: Faint Bands (Weak Signal)

Problem: Extra Bands

Problem: High Background

Problem: Diffuse Bands

Problem: White Bands (ECL method)

Problem: Patchy uneven spots all over the blot