Reactivity	Application
Human	WB (HL-60,U937,Raji,) Western blot analysis of extracts of various cell lines using FGR Polyclonal Antibody at dilution of 1:1000. Western blot analysis of extracts of various cell lines using FGR Polyclonal Antibody at dilution of 1:1000. Western blot analysis of extracts of various cell lines using FGR Polyclonal Antibody at dilution of 1:1000.

Dilution

WB 1:500-1:2000

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Preparation of protein samples

1.Protein extraction

1)For tissue sample
a. Take the samples, wash the tissue thoroughly with pre-cooled PBS (0.01 M, pH=7.4)(Cat# E-BC-R187) to remove the surface blood and internal debris.
b. Weigh and smash the tissue, add an appropriate ratio of RIPA Lysis Buffer (Cat# E-BC-R327)(add 10 μL PMSF and 10 μL Na₃VO₄ to each 1 mL RIPA Lysis) and homogenizely lyse the tissue.
It is recommended to homogenize according to the ratio of tissue weight: RIPA volume = 3:10. For example, add 1 mL RIPA Lysis Buffer to 0.3 g tissue sample, the specific volume can be adjusted according to experimental requirements.
c. Shake and lyse on the ice for 30 min after homogenization. And then sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lysis and reduce the viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2)For cell sample
a. Collect the cells, wash them thoroughly with pre-cooled PBS (0.01 M, pH=7.4) to remove the medium off (it is generally recommended to wash 3 times).
b. Add an appropriate ratio of RIPA Lysate Buffer (10 μL PMSF and 10 μL Na₃VO₄ in each 1 mL RIPA Lysis) and lyse on the ice for 30 min.
It is recommended to add 0.1 mL of RIPA Lysis Buffer to each well of a 6-well plates (the protein content in different cells may vary, and the volume of the lysate added can be appropriately adjusted).
c. Sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lyse and reduce viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2.Measurement of protein concentration
By the BCA method (see the Total Protein Colorimetric Assay Kit (Cat# E-BC-K318) instructions).

3.Boiling the samples
Adjust the protein concentration with PBS Buffer. Add 5 × SDS Loading Buffer (Cat# E-BC-R288) with the ratio of the protein sample: 5 × SDS Loading Buffer = 4:1 and boil the mixture for 10 min. Centrifuge at 12,000 rpm for 2 min and collect the supernatant. The denatured protein can be employed to Western Blot experiments or stored at -20℃ or -80℃.

Note: It is recommended that the total protein loading amount of test sample is about 50 μg in each well. Try to make the loading volume of each sample close to 10 μL.

Electrophoresis

1.According to the molecular weight of the target protein, prepare 0% separation gel. Add the test sample to each well, and add 5 μL of Pre-stained Protein Marker (Cat# E-BC-R273)to a reserved well in order to verify the target molecular weight and the extent of membrane transfer. Add Electrophoresis Buffer ( Cat# E-BC-R331) and start electrophoresis.

2.Electrophoresis at 80v when the samples are in stacking gel, then convert to 120v when the blue flow into the separating gel. Electrophoresis time is about 2-3 h till bromophenol blue reaches the bottom of the gel.

Transfer Membrane

1.Choose the PVDF Membrane (Cat# E-BC-R266) with a pore size of μm according to the molecular weight of the target protein. Soak the PVDF Membrane in methanol for 1 min to activate it, and then soak the PVDF Membrane in the Transmembrane Buffer (Cat# E-BC-R333), the filter paper and fiber mat must be soaked in the Transmembrane Buffer for use too.

2.Follow manufacture instructions of Transfer System for wet, semi-dry, or dry transfer.

Incubation of antibodies

1.Soak the PVDF Membrane with TBST Buffer (Cat# E-BC-R335) containing 5% Skim Milk Powder as blocking buffer and block the membrane at room temperature for .

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the FGR Antibody at , soak the PVDF Membrane in the primary antibody working solution, incubate overnight at 4 ℃, and gently shake.

3.Wash the PVDF Membrane with TBST Buffer for .

4.According to the recommended secondary antibody dilution ratio, use a TBST Buffer solution containing 2% Skim Milk Powder to dilute Goat Anti-Rabbit IgG (H+L) (peroxidase/HRP conjugated) (Cat# E-AB-1003) at . Incubate at room temperature for 1 h on a shaker.

5.Wash the PVDF Membrane with TBST Buffer for .

Detection

1.Mix A and B in the Excellent Chemiluminescent Substrate Detection kit (Cat# E-BC-R347) at the ratio of 1:1 as working solution.

2.Take out the PVDF Membrane from TBST Buffer and absorb the liquid with the filter paper. Pave the PVDF Membrane on the detection machine, add ECL working solution continuously on the PVDF Membrane, discharge the bubble and detect the result.

3.Adjust the contrast and the exposure time to get the best image.

Provided product(E-IR-R304A E-IR-R304B)

Product	Catalog No
RIPA Lysis Buffer (with PMSF and Na₃VO₄)	E-BC-R327
Total Protein Colorimetric Assay Kit (BCA method)	E-BC-K318
Excellent Chemiluminescent Substrate Detection Kit	E-BC-R347
Pre-stained Protein Marker (10-180 kDa)	E-BC-R273
Goat Anti-Rabbit IgG(H+L)-HRP	E-AB-1003
Skim Milk Powder	E-BC-R337
PBS Buffer, pH7.4 (10 ×)	E-BC-R187
5 × SDS Loading Buffer	E-BC-R288
Electrophoresis Buffer (10 ×)	E-BC-R331
Transmembrane Buffer (10 ×)	E-BC-R333
TBST Buffer (10 ×)	E-BC-R335
PVDF Membrane (0.45 μm, 8.5 × 6 cm)	E-BC-R266
PVDF Membrane (0.22 μm, 8.5 × 6 cm)	E-BC-R329

Appendix

Table 1. Preparation of separating gel

Table 2. Preparation of stacking gel

Table 3. Comparison of protein separation linear range

Product Details

Isotype	IgG
Concentration	1mg/mL
Storage	Store at -20°C. Avoid freeze / thaw cycles.
Buffer	PBS with 0.02% sodium azide, 50% glycerol, pH7.3.
Purification Method	Affinity purification
Research Areas	Cancer, Signal Transduction
Conjugation	Unconjugated

Immunogen Details

Immunogen	A synthetic peptide of human FGR (NP_005239.1).
Abbre	FGR
Synonyms	FGR,SRC2,c-fgr,c-src2,p55-Fgr,p55c-fgr,p58-Fgr,p58c-fgr
Swissprot	P09769
Gene ID	2268
Calculated MW	59kDa
Observed MW	53kDa The actual band is not consistent with the expectation. Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. The common factors include: 1. Post-translational modifications: For example, modifications such as glycosylation, phosphorylation, methylation, and acetylation will increase the molecular weight of the protein. 2. Splicing variants: Different expression patterns of various mRNA splicing bodies may produce proteins of different sizes. 3. Post-translational cleavage: Many proteins are first synthesized into precursor proteins and then cleaved to form active forms, such as COL1A1. 4. Relative charge: the composition of amino acids (the proportion of charged amino acids and uncharged amino acids). 5. Formation of multimers: For example, in protein dimer, strong interactions between proteins can cause the bands to be larger. However, the use of reducing conditions can usually avoid the formation of multimers. If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane.
Cellular Localization	Cell membrane. Cell membrane. Cell projection>ruffle membrane. Cytoplasm>cytosol. Cytoplasm>cytoskeleton. Mitochondrion inner membrane. Mitochondrion intermembrane space. Detected in mitochondrial intermembrane space and at inner membranes (By similarity). Colocalizes with actin fibers at membrane ruffles. Detected at plasma membrane lipid rafts.
Tissue Specificity	Detected in neutrophils, monocytes and natural killer cells (at protein level). Detected in monocytes and large lymphocytes.

Related Reagents

Application	Products
WB	Western Blot Detection Kit(E-IR-R304)
IHC	2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System (with DAB solution)(E-IR-R217)

Background

This gene is a member of the Src family of protein tyrosine kinases (PTKs). The encoded protein contains N-terminal sites for myristylation and palmitylation, a PTK domain, and SH2 and SH3 domains which are involved in mediating protein-protein interactions with phosphotyrosine-containing and proline-rich motifs, respectively. The protein localizes to plasma membrane ruffles, and functions as a negative regulator of cell migration and adhesion triggered by the beta-2 integrin signal transduction pathway. Infection with Epstein-Barr virus results in the overexpression of this gene. Multiple alternatively spliced variants, encoding the same protein, have been identified.

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