Thank you for contacting us.
I am sorry to hear that 2 of our antibodies are giving you problems.
For ab104293, regarding the 3 bands in the WB image, I will have to ask the lab for more information. For the WB protocol please see below. At which MW do you see the weak band? According to the UniProt database (http://www.uniprot.org/uniprot/Q8N8V2), no additional isoform or splice variant has been reported.
For ab97935, I am not sure why you see a second band at 40 kDa. Again, according to the UniProt database (http://www.uniprot.org/uniprot/Q9H0R5), no additional isoform or splice variant has been reported.
For both antibodies, could you please let me know:
1) How much protein are you loading?
2) What antibody dilutions are you using?
3) What is the blocking reagent?
4) Is the secondary antibody working well with other primaries?
5) Have you tried any other samples besides THP-1 (e.g. a positive control such as Hela or HepG2 cells)? If so, what were the results there?
Western Blotting Protocol for ab104293:
A. Preparation of cell lysates
1. Collect cells (confluent T-25) by trypsinization and spin.
2. Lyze the pellet with 100 µl lysis buffer on ice for 10 min. For 500,000 cells, lyze with 20 µl.
3. Spin at 14,000 rpm (16,000 g) in microfuge for 10 min at 4°C.
4. Transfer the supernatant to a new tube and discard the pellet.
5. Determine the protein concentration (Bradford assay, A280, or BCA).
6. Take x µl (= y µg protein) and mix with x µl of 2x sample buffer.
7. Boil for 5 min and cool at RT for 5 min.
8. Flash spin to bring down condensation prior to loading gel.
B. Polyacrylamide gel (14.5 cm x 16.5 cm)
1. Agarose plug: 1% agarose dissolved in 1x Resolving gel buffer.
2. Resolving gel: 24 ml of a 9% gel, 5.4 ml 40% acrylamide/bisacrylamide (29:1 mix),
3 ml 8x Resolving gel buffer, 15.6 ml water, 12 µl TEMED, 60 µl 20% ammonium persulfate
3. Stacking gel: 8 ml = 1 ml 40% acrylamide/bisacrylamide (29:1 mix), 2 ml 4x Stacking gel buffer, 5 ml water, 8 µl TEMED, 21.6 µl 20% ammonium persulfate
C. Preparation of gel
1. Assemble the glass plates and spacers (1.5 mm thick).
2. Pour an agarose plug (1-2 mm).
3. Pour the running gel to about 1 cm below the wells of the comb (~20 ml).
4. Seal with 1 ml water-saturated 1-butanol.
5. When gel has set, pour off the butanol and rinse with deionized water.
6. Pour the stacking gel (~5 ml) and insert the comb immediately.
7. When the stacking gel has set, place in gel rig and immerse in buffer.
8. Prior to running the gel, flush the wells out thoroughly with running buffer.
D. Running the gel
1. After flash spinning the samples, load into the wells.
2. Be sure to use markers.
3. Run with constant current (35-37 mA with voltage set at > 150 V).
4. Usual running time is about 1.3 hr.
E. Preparation of membrane
1. Cut a piece of PVDF membrane.
2. Wet in methanol on a rocker at RT for 5 mins. Remove methanol and add 1x Transfer buffer until ready to use.
F. Membrane transfer
1. Assemble "sandwich" for Bio-Rad's Transblot.
2. Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer.
Sponge - filter paper - gel - membrane - filter paper - sponge
3. Transfer for 1 hr at 15 volts at 4°C on a stir plate. Bigger proteins might take longer to transfer.
For the Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer.
4. Immerse membrane in Amido-Black stain 5 mins.
5. Destain 4 x 5 mins with destaining buffer.
6. When finished, immerse membrane in Blocking buffer and block for one hour at room temperature.
G. Antibodies and detection
1. Incubate with primary antibody diluted to 2 ug/ml in total volume of 3 mL in Blocking buffer for one hour at room temperature.
2. Wash 4 x 5 min with 0.05% Tween 20 in TBS.
3. Incubate with secondary antibody diluted 1:10,000 (HRP anti-rabbit) in Blocking buffer for 1 hour at room temp.
4. Wash 4 x 5 min with 0.05% Tween 20 in TBS.
5. Detect with Chemiluminescent kit.
H. Stripping blot
1. Rinse blot off with 0.05% Tween 20 in PBS.
2. Put blot into Kapak bag cut to slightly bigger size than blot.
3. Add about 5 to 10 ml Stripping buffer.
4. Remove as much air as possible and seal bag.
5. Immerse into 80°C water bath and incubate for 20 min.
6. Rinse blot off with 0.05% Tween 20 in PBS.
7. Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween20.
Buffers for Westerns
0.15 M NaCl
5 mM EDTA, pH 8
1% Triton X100
10 mM Tris-Cl, pH 7.4
Just before using add:
1:1000 5 M DTT
1:1000 100 mM PMSF in isopropanol
1:1000 5 M e-aminocaproic acid
2x sample buffer:
130 mM Tris-Cl, pH8.0
20% (v/v) Glycerol
4.6% (w/v) SDS
0.02% Bromophenol blue
8x Resolving gel buffer: 100 ml
0.8 g SDS (add last)
36.3 g Trizma base (= 3 M)
Adjust pH to 8.8 with concentrated HCl
4x Stacking gel buffer: 100 ml
0.4 g SDS (add last)
6.05 g Trizma base (= 0.5 M)
Adjust pH to 6.8
10x Running buffer: 1 L
30.3 g Trizma base (= 0.25 M)
144 g Glycine (= 1.92 M)
10 g SDS (= 1%)--add last
Do not adjust the pH!!
10x Blotting buffer: 1 L
30.3 g Trizma base (= 0.25 M)
144 g Glycine (= 1.92 M)
pH should be 8.3; do not adjust
To make 2 L of 1x Blotting buffer:
400 ml Methanol
200 ml 10x Blotting buffer
1400 ml water
Blocking buffer: 0.5 L
3% Bovine serum albumin (Fraction V)
Make up in PBS and sterile filter. Then add 0.05% Tween 20. Keep at 4°C to prevent bacterial contamination.
0.5 L (sterile filter solution and keep at 4°C)
0.2 M Glycine, pH 2.5
0.05% Tween 20
I look forward to hear back from you.