Silver staining protein gels (Stukenberg lab) and Immunofluorescence with biotinylated antibodies (Stukenberg lab): Difference between pages

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Silver Staining Protocol (Not Compatible with Mass Spec)


Immunofluorescence with Biotinylated Antibodies


Notes:
Be careful not to touch gel during any step of the procedure with either a gloved or ungloved hand, finger etc…
Use MilliQ-H2O for all steps
Perform all incubations in well washed glass dishes.
   
   
 
1. Fix cells onto coverslips as normal, and wash appropriately.
 
1. Remove gels from glass plates in which they were run:
2. Add to your normal first block (20% serum, or 3%BSA, etc., in your buffer of interest; I use PBST or TBST depending on the antibody), approximately 10ul/100ul of Avidin blocking solution (Vector Laboratories, catalog # SP-2001)
Block as usual, ~30 minutes at room temperature.
3. Incubate your coverslips in your 1st rabbit primary antibody as usual, (5% serum in your chosen buffer, etc.) at room temperature for 1hour.
   
   
2. Incubate in 50% Methanol, 10% Glacial Acetic Acid, 40% H2O for 30 minutes (100 ml)
4. Wash the coverslips twice for 5 minutes each in PBST or your buffer of choice.  I rock my coverslips gently in 6 or 12 well dishes on a rotator.
   
   
3. Incubate in 5% Methanol, 7% Acetic Acid (Glacial), 88% H2O for 30 minutes (100ml)
5. Add 10uL/100uL of Biotin blocking solution to the 1st anti-rabbit 2? antibody, and label the coverslips as usual (5% serum in your chosen buffer), ~45 minutes at room temperature in the dark to protect the fluorophore.
   
   
4. Incubate in 10% Gluteraldehyde made up in H2O from 25% stock (25 ml) for 30 minutes.  
6. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer.  Keep the coverslips covered (I use some foil) to protect your fluorophore.
   
   
5. Wash 4 x 20-30 minutes in H2O using 200-300 ml per wash.
7. This is the critical step: Block the coverslips in rabbit serum or IgG.  This will block any leftover IgG from the first anti-rabbit secondary antibody that might be recognized by the biotinylated rabbit antibody that you will use in the next step.  If you forget this step, your results will be skewed significantly by the high background staining resulting from the biotinylated rabbit antibody binding to your anti-rabbit secondary used in the previous step.
   
   
6. Makes 2 Solutions, A and B:    A) 3 ml of 30% Ammonia Hydroxide, 0.25 g NaOH in 190 ml H2O.  B) 1.2g Silver Nitrate (AgNO3) into 10 ml of H2O. 
8. Probe the coverslips with your biotinylated 2nd primary rabbit antibody (5% serum in buffer), 1 hour at room temperature in the dark.
   
   
7. Add solution B to A slowly, mix gently, and add to gel, incubate for 30 minutes.
9. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer.  Keep the coverslips covered to protect your fluorophore.
   
   
8. Make developer and stop solutions (see below).
10. Incubate the coverslips with Avidin-Cy3 or Avidin-FITC as your 2nd rabbit antibody secondary, for ~45 minutes at room temperature in the dark.  (I generally use Avidin-Cy3 (Sigma E-4142) at 1:5,000).
   
   
9. Perform 5 x 1 minute washes with H2O, using 200 ml’s per wash.
11. Wash coverslips as before, then wash coverslips 1X with ddH2O.
   
   
10. Add developer solution (~100 ml) and wait and watch gel until you see bands come up. Quickly remove developer solution and add stop solution.  (Developer Solution: 0.1g Citric Acid, 1ml 37% formaldehyde into 1L H2O. Stop Solution: 190 ml H2O and 10 ml Acetic Acid, Glacial)
13. Stain coverslips with DAPI or Hoescht 33342.
   
   
11. After 10 minutes in Stop Solution wash thoroughly
14. Mount coverslips with mounting media onto slides and seal them.

Revision as of 10:54, 14 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


Immunofluorescence with Biotinylated Antibodies


1. Fix cells onto coverslips as normal, and wash appropriately.

2. Add to your normal first block (20% serum, or 3%BSA, etc., in your buffer of interest; I use PBST or TBST depending on the antibody), approximately 10ul/100ul of Avidin blocking solution (Vector Laboratories, catalog # SP-2001) Block as usual, ~30 minutes at room temperature.

3. Incubate your coverslips in your 1st rabbit primary antibody as usual, (5% serum in your chosen buffer, etc.) at room temperature for 1hour.

4. Wash the coverslips twice for 5 minutes each in PBST or your buffer of choice. I rock my coverslips gently in 6 or 12 well dishes on a rotator.

5. Add 10uL/100uL of Biotin blocking solution to the 1st anti-rabbit 2? antibody, and label the coverslips as usual (5% serum in your chosen buffer), ~45 minutes at room temperature in the dark to protect the fluorophore.

6. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer. Keep the coverslips covered (I use some foil) to protect your fluorophore.

7. This is the critical step: Block the coverslips in rabbit serum or IgG. This will block any leftover IgG from the first anti-rabbit secondary antibody that might be recognized by the biotinylated rabbit antibody that you will use in the next step. If you forget this step, your results will be skewed significantly by the high background staining resulting from the biotinylated rabbit antibody binding to your anti-rabbit secondary used in the previous step.

8. Probe the coverslips with your biotinylated 2nd primary rabbit antibody (5% serum in buffer), 1 hour at room temperature in the dark.

9. Wash coverslips twice for 5 minutes each in PBST or your chosen buffer. Keep the coverslips covered to protect your fluorophore.

10. Incubate the coverslips with Avidin-Cy3 or Avidin-FITC as your 2nd rabbit antibody secondary, for ~45 minutes at room temperature in the dark. (I generally use Avidin-Cy3 (Sigma E-4142) at 1:5,000).

11. Wash coverslips as before, then wash coverslips 1X with ddH2O.

13. Stain coverslips with DAPI or Hoescht 33342.

14. Mount coverslips with mounting media onto slides and seal them.

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